PACAP deficient mice display reduced carbohydrate intake and PACAP activates NPY-containing neurons in the rat hypothalamic arcuate nucleus

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates both insulin release from islets and insulin action in adipocytes. Therefore, this peptide is considered a regulator of glucose homeostasis. PACAP and its receptors are localized not only in the peripheral tissues but in the central nervous system. The present study examined whether PACAP regulates the feeding behavior and the activity of neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. Food intake was measured in the PACAP knock-out mice. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single neurons isolated from the ARC of rats was measured by fura-2 microfluorometry, followed by immunocytochemical staining with anti-NPY antiserum. PACAP knock-out mice showed a decrease in the intake of high carbohydrate, but not high fat, food. PACAP increased [Ca²⁺]_i in NPY neurons of the ARC that are implicated in the feeding, particularly the carbohydrate ingestion. Agonists of PACAP receptors, PAC1-R and VPAC2-R, also increased [Ca²⁺]_i. The present study, by demonstrating that PACAP directly reacts with the ARC NPY neurons to increase [Ca²⁺]_i and that ingestion of the carbohydrate-rich food is reduced in PACAP-deficiency, suggests a facilitative role for PACAP in the carbohydrate intake.     

Evidence for the involvement of VPAC1 and VPAC2 receptors in pressure-induced vasodilatation in rodents

A transient increase in skin blood flow in response to an innocuous local pressure application, defined as pressure-induced vasodilatation (PIV), delays the occurrence of ischaemia, suggesting a protective feature against applied pressure. The PIV response depends on capsaicin-sensitive nerve fibres and calcitonin gene-related peptide (CGRP) has been shown to be involved. In these fibres, CGRP coexists with pituitary adenylate cyclase-activating polypeptide (PACAP). Three distinct receptors mediate the biological effects of PACAP: VPAC1 and VPAC2 receptors binding with the same affinity for PACAP and vasoactive intestinal peptide and PAC1 receptors showing high selectivity for PACAP. Because the receptors are widely expressed in the nervous system and in the skin, we hypothesized that at least one of them is involved in PIV development. To verify this hypothesis, we used [ d - p -Cl-Phe⁶,Leu¹⁷]-VIP (nonspecific antagonist of VPAC1/VPAC2 receptors), PG 97-269 (antagonist of VPAC1 receptors), PACAP(6–38) (antagonist of VPAC2/PAC1 receptors) and Max.d.4 (antagonist of PAC1 receptors) in anaesthetized rodents. The blockade of VPAC1/VPAC2, VPAC1 or VPAC2/PAC1 receptors eliminated the PIV response, whereas PAC1 blockade had no effect, demonstrating an involvement of VPAC1/VPAC2 receptors in PIV development. Moreover, endothelium-independent and -dependent vasodilator responses were unchanged by the VPAC1/VPAC2 antagonist. Thus, the absence of a PIV response following VPAC1/VPAC2 blockade cannot be explained by any dysfunction of the vascular smooth muscle or endothelial vasodilator capacity. The involvement of VPAC1/VPAC2 receptors in the development of PIV seems to imply a series relationship in which each receptor type (CGRP, VPAC1, VPAC2) is necessary for the full transmission of the response.     

Analysis of the role of the PAC1 receptor in neutrophil recruitment, acute-phase response, and nitric oxide production in septic shock

Infections caused by Gram-negative bacteria constitute one of the major causes of septic shock, which results from the inability of the immune system to limit bacterial spread during the ongoing infection. In the last decade, it has been demonstrated that vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two endogenous immunopeptides, which together with three G protein-coupled receptors (VPAC1, VPAC2, and PAC1) exert a significant, therapeutic effect attenuating the deleterious consequences of septic shock by balancing pro- and anti-inflammatory factors. We have recently shown PAC1 receptor involvement in vivo as an anti-inflammatory receptor, at least in part, by attenuating lipopolysaccharide-induced production of proinflammatory interleukin-6. The present study deepens in the protective role of PAC1 receptor in septic shock, elucidating its involvement in the modulation of neutrophil recruitment and in the expression of different molecular sensors such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, fibrinogen, serum amyloid A, and nitric oxide as important, systemic players of the development of septic shock. Our results, using a mice deficient in PAC1 and a PAC1 antagonist, show that VIP and PACAP as well as the PAC1 receptor are involved in neutrophil recruitment in different target organs, in adhesion molecules expression, and in coagulation-related molecule fibrinogen synthesis. Thus, this study provides some important insights with respect to the involvement of PAC1 into the complexities of sepsis and represents an advantage for the design of more specific drugs complementing standard intensive care therapy in severe sepsis, confirming VIP and PACAP as candidates for multitarget therapy of septic shock.     

PACAP modulation of the colon–inferior mesenteric ganglion reflex in the guinea pig

We investigated the effect of pituitary adenylate cyclase activating peptide (PACAP) on the colon–inferior mesenteric ganglion (IMG) reflex loop in vitro . PACAP27 and PACAP38 applied to the IMG caused a prolonged depolarization and intense generation of fast EPSPs and action potentials in IMG neurones. Activation of PACAP-preferring receptors (PAC1-Rs) with the selective agonist maxadilan or vasoactive intestinal peptide (VIP)/PACAP (VPAC) receptors with VIP produced similar effects whereas prior incubation of the IMG with selective PAC1-R antagonists PACAP6-38 and M65 inhibited the effects of PACAP. Colonic distension evoked a slow EPSP in IMG neurones that was reduced in amplitude by prolonged superfusion of the IMG with either PACAP27, maxidilan, PACAP6-38, M65 or VIP. Activation of IMG neurones by PACAP27 or maxadilan resulted in an inhibition of ongoing spontaneous colonic contractions. PACAP-LI was detected in nerve trunks attached to the IMG and in varicosities surrounding IMG neurones. Cell bodies with PACAP-LI were present in lumbar 2–3 dorsal root ganglia and in colonic myenteric ganglia. Colonic distension evoked release of PACAP peptides in the IMG as measured by radioimmunoassay. Volume reconstructed images showed that a majority of PACAP-LI, VIP-LI and VAChT-LI nerve endings making putative synaptic contact onto IMG neurones and a majority of putative receptor sites containing PAC1-R-LI and nAChR-LI on the neurones were distributed along secondary and tertiary dendrites. These results suggest involvement of a PACAP-ergic pathway, operated through PAC1-Rs, in controlling the colon–IMG reflex.     

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.     

PAC1 receptors in chick cerebral cortex: characterization by binding of pituitary adenylate cyclase-activating polypeptide, [125I]-PACAP27

In this study we characterized receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) in chick cerebral cortex by in vitro binding technique, using [125I]-PACAP27 as a ligand. The specific binding of [125I]-PACAP27 to chick cerebral cortical membranes was found to be rapid, stable, saturable, and of high affinity. Scatchard analysis suggested binding to a single class of receptor binding sites with high affinity (K(d)=0.41+/-0.08 nM) and high capacity (B(max)=457+/-35 fmol/mg protein). The relative rank order of potency of the tested peptides to inhibit [125I]-PACAP27 binding to chick cerebrum was: PACAP38 approximately PACAP27>PACAP6-27 approximately PACAP6-38 > chicken VIP > mammalian VIP > secretin (inactive). It is concluded that the cerebral cortex of chick, in addition to VPAC recognition sites, contains a large population of PAC(1)-type receptor binding sites.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the brain

Recent progress in research on pituitary adenylate-activating polypeptide (PACAP) with a special emphasis on the brain is reviewed. PACAP is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide family. PACAP functions as a hypothalamic hormone, neurotransmitter, neuromodulator, and neurotrophic factor. Studies on the gene encoding the PACAP precursor and the specific PACAP receptor (PAC1-R) and its subtypes have provided information on the control of gene expression for PACAP, and the relationship between the receptor subtypes and the signal transduction pathways. The PAC1-R is a G protein-coupled receptor with seven transmembrane domains and belongs to the VIP receptor family. At least eight subtypes of PAC1-R result from alternate splicing. Each subtype is coupled to specific signaling pathways, and its expression is tissue or cell specific. PACAP stimulates the release of arginine vasopressin and increases cytosolic Ca2+ ([Ca2+]i). PACAP serves as a neurotransmitter and/or neuromodulator and the activation of the PAC1-R stimulates a cAMP-protein kinase A signal transduction pathway which in turn evokes the [Ca2+]i signaling system. More importantly, PACAP is a neurotrophic factor that may play an important role during the development of the brain. The PAC1-R is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development. In the adult brain, PACAP appears to function as a neuroprotective factor that attenuates the neuronal damage resulting from various insults.     

PACAP up-regulates the expression of apolipoprotein D in 3T3-L1 adipocytes. DRG/3T3-L1 co-cultures study

The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation.     

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic AMP formation in the duck and goose brain

Two molecular forms of pituitary adenylate cyclase-activating polypeptide (PACAP), i.e., PACAP27 and PACAP38 (0.0001-1 microM), as well as vasoactive intestinal polypeptide (VIP; 0.1-3 microM), have been studied for their effects on cyclic AMP formation in the hypothalamus and cerebral cortex of duck and goose. All three peptides concentration-dependently stimulated cyclic AMP production in the tested brain regions of 2-3-weeks-old (young) ducks, with VIP showing at least one order of magnitude weaker activity than PACAP. This characteristics suggests the existence in the duck's brain of adenylyl cyclase-linked PAC1 receptors. Both forms of PACAP also stimulated the nucleotide formation in the cerebral cortex and hypothalamus of 5-6-months-old (adult) ducks or geese grown under natural environment. The peptides-evoked effects in adult and young ducks were comparable, and clearly greater than those found in adult geese. The present data extend our recent observations made on chicks, and suggest PACAP to be a potent stimulator of the cyclic AMP generation in the avian central nervous system.     

Excitatory actions of vasoactive intestinal peptide on mouse thalamocortical neurons are mediated by VPAC2 receptors

Thalamic nuclei can generate intrathalamic rhythms similar to those observed at various arousal levels and pathophysiological conditions such as absence epilepsy. These rhythmic activities can be altered by a variety of neuromodulators that arise from brain stem regions as well as those that are intrinsic to the thalamic circuitry. Vasoactive intestinal peptide (VIP) is a neuropeptide localized within the thalamus and strongly attenuates intrathalamic rhythms via an unidentified receptor subtype. We have used transgenic mice lacking a specific VIP receptor, VPAC(2), to identify its role in VIP-mediated actions in the thalamus. VIP strongly attenuated both the slow, 2-4 Hz and spindle-like 5-8 Hz rhythmic activities in slices from wild-type mice (VPAC(2)(+/+)) but not in slices from VPAC(2) receptor knock-out mice (VPAC(2)(-/-)), which suggests a major role of VPAC(2) receptors in the antioscillatory actions of VIP. Intracellular recordings revealed that VIP depolarized all relay neurons tested from VPAC(2)(+/+) mice. In VPAC(2)(-/-) mice, however, VIP produced no membrane depolarization in 80% of neurons tested. In relay neurons from VPAC(2)+/+ mice, VIP enhanced the hyperpolarization-activated mixed cation current, I(h), via cyclic AMP activity, but VIP did not alter I(h) in VPAC(2)-/- mice. In VPAC(2)-/- mice, pituitary adenylate cyclase activating-polypeptide (PACAP) depolarized the majority of relay neurons via I(h) enhancement presumably via PAC(1) receptor activation. Our findings suggest that VIP-mediated actions are predominantly mediated by VPAC(2) receptors, but PAC(1) receptors may play a minor role. The excitatory actions of VIP and PACAP suggest these peptides may not only regulate intrathalamic rhythmic activities, but also may influence information transfer through thalamocortical circuits.     

Reduction of lipopolysaccharide-induced neurotoxicity in mixed cortical neuron/glia cultures by femtomolar concentrations of pituitary adenylate cyclase-activating polypeptide.

Stimulation of murine primary mixed cortical neuron/glia cultures with lipopolysaccharide, an endotoxin, was used as a model for inflammatory disorders of the central nervous system. Lipopolysaccharide (20 microg/ml) increased the secretion of lactate dehydrogenase, a marker for cell injury, and nitric oxide into the culture medium. The lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium was reduced by pituitary adenylate cyclase-activating polypeptide (PACAP) at 10(-14)-10(-12) M. The 27- and 38-amino-acid forms of PACAP were equipotent and their dose-response curves were U-shaped. PACAP6-38, a specific type I PACAP receptor antagonist, blocked the reduction by PACAP38 of the lipopolysaccharide-induced release of lactate dehydrogenase. The lipopolysaccharide-induced secretion of nitric oxide into the culture medium was reduced by PACAP at 10(-14)-10(-12) M and 10(-8)-10(-6) M. The 27- and 38-amino-acid forms of PACAP were equipotent. PACAP6-38 blocked the reduction of the lipopolysaccharide-induced secretion of nitric oxide by PACAP38 at 10(-12) M, but not at 10(-8) M. Vasoactive intestinal polypeptide reduced the lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium at 10(-14)-10(-12) M, but these concentrations of vasoactive intestinal polypeptide had no effect on the lipopolysaccharide-induced secretion of nitric oxide. PACAP6-38 did not effect the reduction of the lipopolysaccharide-induced release of lactate dehydrogenase into the culture medium by 10(-12) M vasoactive intestinal polypeptide. These results indicate that stimulation of type I PACAP receptors by femtomolar concentrations of PACAP can prevent neuron death in a model for inflammatory disorders of the CNS. These results suggest that PACAP is also an extraordinarily potent inhibitor of some microglial functions.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an upstream regulator of prodynorphin mRNA expression in neurons

Although dynorphins are widely involved in the control of not only nociceptive neurotransmission but also a variety of brain functions such as memory and emotion, no natural regulator for inducing the mRNA expression of prodynorphin (Pdyn), a precursor protein of dynorphins, is known. Using primary cultures of rat cortical neurons, we found that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon neuropeptide family, markedly induces Pdyn mRNA expression. PACAP was much more effective than VIP, indicating a major role for PAC1 in the PACAP-induced Pdyn mRNA expression. The increase in Pdyn mRNA expression was independent of de novo protein synthesis. Administration of forskolin, an activator for adenylate cyclase/protein kinase A (PKA), but not TPA, an activator for protein kinase C (PKC), induced Pdyn mRNA expression, suggesting a major role for PKA. The involvement of PKA was supported by the inhibition of PACAP-induced Pdyn mRNA expression upon addition of H89, an inhibitor for PKA. The PACAP-induced potentiation of NMDA-R was involved in the mRNA expression of Bdnf or c-fos but not Pdyn. These results suggest PACAP to be an upstream regulator for inducing Pdyn mRNA expression through PKA.     

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.