Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (VIP)

Molecular identification of the binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and the effect of vasoactive intestinal peptide (VIP) on the specific binding sites for PACAP in rat cultured astrocyte membrane preparations were investigated. Affinity cross-linking of astrocyte membrane preparations with [125I]PACAP27 showed the presence of a 60 kDa radiolabeled ligand-receptor complex. The labeling of this band was completely abolished in the presence of 10(-8) M or higher concentrations of unlabeled PACAP27. The molecular weight of this binding protein was estimated to be 57 kDa assuming an equimolar interaction of ligand and receptor in the 60 kDa complex. The labeling of [125I]PACAP27 binding to this binding protein was partly reduced by the addition of 10(-6) M VIP, but not by 10(-8) M. In the binding assay, VIP displaced the specific binding of [125I]PACAP27 at 10(-7) M or a greater concentration. Displacement of [125I]PACAP27 binding by unlabeled PACAP27 was analyzed in the presence or absence of 10(-6) M VIP. VIP at 10(-6) M reduced the maximal binding capacity (Bmax) of the high affinity binding site for PACAP27 by about 50% but did not alter the Bmax of the low affinity binding site. The dissociation constants (Kd) for both the high and low affinity binding sites were unaltered. These results indicate that PACAP binds to a 57 kDa membrane protein with high affinity and that VIP, at much higher concentrations, binds to this same binding site, suggesting that VIP mimics the biological action of PACAP in astrocytes at high concentrations.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on the cardiovascular system in sheep.

The cardiovascular effects of PACAP and VIP were studied in intact conscious sheep; PACAP (0.008, 0.04, 0.2, and 1.0 nmol/min) and VIP (0.07, 0.2, 0.6, and 1.8 nmol/min) were infused in conscious sheep for periods of 10 min. For each peptide there was a dose-dependent increase in heart rate. At the highest doses tested, pulse pressure and mean arterial pressure tended to increase and decrease, respectively. However, only the decrease in mean arterial pressure following the highest dose of VIP reached significance. At the highest doses tested, heart rate increased nearly threefold during the infusion while mean arterial pressure declined by 18.5%. In individual animals the decrease in blood pressure and increase in heart rate occurred simultaneously, so that we were unable to conclude whether the increase in heart rate was due to a baroreceptor reflex.     

The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes.

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.     

Vascular effects of pituitary adenylate cyclase activating peptide: a comparison with vasoactive intestinal peptide

The effects of pituitary adenylate cyclase activating peptide (PACAP) on the blood pressure of the anesthetized rat and on the isolated rat tail artery were investigated and compared to those of vasoactive intestinal peptide (VIP). PACAP-38, PACAP-27 and the C-terminal fragment 16–38 caused a dose-dependent decrease in the systemic blood pressure. PACAP-27 and PACAP-38 were equipotent with VIP. The C-terminal fragment 16–38 was much less potent than VIP. The duration of action was longer for equimolar doses of PACAP-38 and PACAP-27 than for VIP and much longer than for PACAP 16–38. PACAP-27 and the phosphodiesterase inhibitor rolipram given in combination produced additive vasodepressive responses. In vitro PACAP-38, PACAP-27, VIP and PACAP 16–38 relaxed the phenylephrine-precontracted rat tail artery. PACAP-38 and PACAP-27 were equipotent with VIP. PACAP 16–38 was much less potent than the full-length peptides. The responses were resistant to atropine and propranolol. Addition of VIP 1 μM to preparations exposed to 1 μM PACAP-38 or -27 did not produce a further relaxation. VIP-like peptides, PACAP in particular, are known to activate adenylate cyclase and to elevate the plasma cyclic AMP (cAMP) concentration. cAMP was found to be a potent vasodepressor in the anaesthetized rat and a potent vasodilator of precontracted blood vessels. On the basis of these results it cannot be excluded that the vascular effects of PACAP are secondary to the effect of elevated levels of extracellular cAMP.     

垂体腺苷酸环化酶激活肽的发现及研究进展

垂体腺苷酸环化酶激活肽是最初在绵羊下丘脑发现的一种新的具有多种生物活性的多肽。它广泛分布于中枢神经系统、周围神经系统以及非神经组织内。此外,它在某些类型细胞的旁分泌和自分泌主财节中也发挥作用。     

Investigation of pituitary adenylate cyclase activating polypeptide (PACAP) in human amniotic fluid samples

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide acting as a hormone, a neuromodulator, a neurotransmitter, a trophic factor and is involved in a variety of developmental and regenerative processes. PACAP is present in several human tissues and biological fluids. In many pathological conditions, changes in PACAP levels have been described to reflect disease progression, therefore PACAP has diagnostic value as a potential biomarker. Since PACAP has been shown to play an important role in reproductive physiology and development, it was of interest to examine whether this neuropeptide occurs in the human amniotic fluid. Amniotic fluid samples were collected between the 15-19^th weeks of gestation from volunteering pregnant women undergoing amniocentesis as a prenatal diagnostic tool due to maternal age. Pathological cases were excluded after prenatal karyotype analysis. PACAP-like immunoreactivity was measured by radioimmunoassay and could be detected in all samples. The present study provides evidence for the presence of PACAP in human amniotic fluid, but determination of the exact physiological or pathological significance awaits further investigation.     

Vasoactive intestinal polypeptide (VIP) stimulates adenylate cyclase in selected areas of rat brain

VIP stimulated adenylate cyclase activity in homogenates of some areas of rat brain that are rich in this peptide, e.g., cerebral cortex, hypothalamus and hippocampus, as well as in cerebellar cortex, where VIP content is low. No stimulation occurred in caudate nucleus or brainstem. The enzyme stimulation was inhibited by Ca²⁺, but unaffected by guanine nucleotides. Synthetic fragments of VIP (VIP_6?28 & VIP_14–28) neither stimulated cyclase activity nor inhibited VIP-induced stimulation.     

Properties and distribution of receptors for pituitary adenylate cyclase activating peptide (PACAP) in rat brain and spinal cord.

A high density (in the pmol/mg protein range) of specific functional receptors for PACAP (pituitary adenylate cyclase activating polypeptide) was observed in membranes from rat brain cortex, olfactory bulb, hypothalamus, hippocampus, striatum, cerebellum, pons and cervico-dorsal spinal cord, using [125I]PACAP-27 (PACAP 1-27). The tracer bound rapidly, specifically and reversibly. Competition binding curves were compatible with the coexistence, in the eight central nervous areas explored, of high and low affinity binding sites for PACAP-27 (Kd of 0.2 nM and 3.0 nM, respectively), and of only one class of binding sites for PACAP-38 (PACAP (1-38), Kd 0.2-0.9 nM). VIP inhibited only partially the binding of [125I]PACAP-27, and PHI, GRF(1-29)NH2 and secretin were ineffective at 1 microM. Chemical [125I]PACAP-27 cross-linking revealed a single specific 64 kDa protein species. In rat brain cortical membranes, saturation and competition experiments, using [125I]PACAP-38 as radioligand, indicated the presence of both high (Kd 0.13 nM) and low (Kd 8-10 nM) affinity binding sites for PACAP-38 and of low affinity (Kd 30 nM) binding sites for PACAP-27. These data taken collectively suggest the coexistence of PACAP-A receptors with a slight preference for PACAP-27 over PACAP-38 and of PACAP-B receptors that recognize PACAP-38 with a high affinity and PACAP-27 with low affinity. Both PACAP-27 and PACAP-38 stimulated adenylate cyclase with similar potency and efficacy. VIP was markedly less potent in this respect and also less efficient, except on cerebellar membranes.     

Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity in the ureter of the duck

The presence, distribution and colocalisation of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity have been studied in the duck ureter by using Western blot analysis, radioimmunoassays (RIA) and immunohistochemistry. The presence of both PACAP-38 and PACAP-27 was demonstrated, PACAP-38 being the predominant form. PACAP-immunoreactive fibres and neurons were found in all the ureteral layers. Double immunostaining showed that PACAP was almost completely colocalised with vasoactive intestinal peptide (VIP). Moreover, PACAP was found in substance P (SP)-containing ureteral nerve fibres and in SP-containing dorsal root ganglion neurons. RIA performed on denervated ureters demonstrated that almost half of the ureteral PACAP was extrinsic in origin. These findings suggest that, in birds, PACAP has a role in diverse nerve-mediated ureteral functions.     

Vasoactive intestinal peptide (VIP): an amnestic neuropeptide

Vasoactive intestinal peptide (VIP) is a neuropeptide present in high concentrations in the hippocampus. The studies reported here demonstrate that VIP administered into the third ventricle of the brain caused amnesia in mice trained on a left-right footshock avoidance task in a T-maze. VIP resulted in amnesia when administered directly into the rostral portion of the hippocampus at a 10-fold lower dose than was needed to produce amnesia when VIP was administered intracerebroventricularly. When VIP was administered 24 hr after training, it failed to impair retention measured a week later. VIP receptor antagonist ([4-Cl-D-Phe6,Leu17]VIP) enhanced retention when administered into the rostral portion of the hippocampus, suggesting that VIP plays a physiological role in memory modulation. VIP receptor antagonist administered 24 hr after training did not facilitate retention. To gain some insight as to how VIP may be affecting memory processing, we determined if some memory-improving compounds showed a selective ability to block amnesia induced by VIP. The amnestic effect of VIP was blocked by peripheral administration of the memory-enhancing agents, arecoline, naloxone and ST 587 (a noradrenergic receptor agonist) but not by cholecystokinin octapeptide. Central administration of arecoline, but not neuropeptide Y, blocked the amnestic effect of VIP. It is concluded that VIP is a potent amnestic peptide.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

In vivo effect of pituitary adenylate cyclase activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats.

In view of the recent demonstrations that pituitary adenylate cyclase activating polypeptide (PACAP) 38 stimulates the release of LH from superfused pituitary cells and that the hypothalamus and anterior pituitary have highly selective binding sites for the peptide, we have surveyed the effect of intraatrial injections of PACAP 38 and vasoactive intestinal peptide (VIP), which has 68% homology with PACAP 38, in intact adult male rats. Furthermore the effect of intracerebroventricular (icv) injection of PACAP 38 was investigated. Intraatrial (10, 30, 100 micrograms) and icv (8, 32 micrograms) administration of PACAP 38 stimulated LH release significantly (P less than 0.01) in a dose-related fashion. Icv injection at a dose of 0.8 microgram was ineffective. The time course pattern of LH release by intraatrial injection and that by icv injection was similar, but the LH levels increased by intraatrial injection were much higher than that by icv injection. Intraatrial administration of VIP had almost no effect on LH release. These findings suggested that PACAP 38 stimulates LH release in vivo.     

Anti-shock effect of pituitary adenylate cyclase activating polypeptide (PACAP) on experimental endotoxin shock in dogs

Effects of pituitary adenylate cyclase activating polypeptide with 38 amino acid residues (PACAP-38) on both cardiovascular functions and plasma hormone levels during endotoxin shock were studied in anesthesized dogs. When PACAP-38 (a bolus 420 pmol/kg injection or a bolus 420 pmol/kg injection followed by a continuous 30 pmol/kg/min infusion for 60 min) was administered intravenously 5 min after application of endotoxin, both mean arterial pressure and cardiac output were restored at 10 min. The continuous administration of PACAP-38 was more effective in improving the symptoms of shock. Plasma adrenalin and cortisol levels were significantly increased by both regimens. These results clearly indicate that the anti-shock properties of PACAP-38 may be attributed to its abilities to increase plasma cortisol and adrenalin levels and to stimulate cardiac function.     

Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

Summary Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.