Pituitary Adenylate Cyclase-activating Peptide Type Research Articles

PACAP6-38 improves nitroglycerin-induced central sensitization by modulating synaptic plasticity at the trigeminal nucleus caudalis in a male rat model of chronic migraine

AimsChronic migraine (CM) is a common neurological disorder with complex pathogenesis. Evidence suggests that pituitary adenylate cyclase-activating peptide (PACAP) induces migraine-like attacks and may be potential a new target for migraine treatment, but the therapeutic results of targeting PACAP and its receptors are not uniform. Therefore, the aim of this study was to investigate the regulatory effect of PACAP type I receptor (PAC1R) antagonist, PACAP6-38, on nitroglycerin (NTG)-induced central sensitization in a CM model.MethodsSprague–Dawley (SD) rats received repeated injections of NTG to construct a CM model. Mechanical and thermal thresholds were measured using Von Frey filaments and hot plate tests. C-Fos expression was measured by western blotting and immunofluorescence staining to assess the central sensitization. PACAP6-38 was intracerebrally injected into the trigeminal nucleus caudalis (TNC), and then the changes in c-Fos, the synaptic-associated proteins, phospho-ERK1/2 (p-ERK1/2), phosphorylation of cyclic adenosine monophosphate response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) were detected. Transmission electron microscopy (TEM) and Golgi-Cox staining were used to observe the ultrastructure of synapses and dendritic structures of TNC neurons.ResultsThe results showed that PACAP and PAC1R expression were significantly raised in the TNC after repeated NTG injections. Additionally, PACAP6-38 treatment alleviated nociceptive sensitization, inhibited NTG-induced overexpression of c-Fos and synaptic-associated proteins in the TNC of CM rat, restored aberrant synaptic structures. Furthermore, the expression of ERK/CREB/BDNF pathway was depressed by PACAP6-38.ConclusionsOur results demonstrated that abnormal synaptic structure in the TNC of CM, which could be reversed by inhibition of PAC1R via down-regulating the ERK/CREB/BDNF signaling pathway. PACAP6-38 improves NTG-induced central sensitization by regulating synaptic plasticity in the TNC of CM rat, which may provide new insights into the treatments targeting PACAP/PAC1R in migraine.

Peptide OM-LV20 protects astrocytes against oxidative stress via the ‘PAC1R/JNK/TPH1’ axis

Stroke can lead to severe nerve injury and debilitation, resulting in considerable social and economic burdens. Due to the high complexity of post-injury repair mechanisms, drugs approved for use in stroke are extremely scarce, and thus, the discovery of new antistroke drugs and targets is critical. Tryptophan hydroxylase 1 (TPH1) is involved in a variety of mental and neurobehavioral processes, but its effects on stroke have not yet been reported. Here, we used primary astrocyte culture, quantitative real-time PCR, double immunofluorescence assay, lentiviral infection, cell viability analysis, Western blotting, and other biochemical experiments to explore the protective mechanism of peptide OM-LV20, which previously exhibited neuroprotective effects in rats after ischemic stroke via a mechanism that may involve TPH1. First, we showed that TPH1 was expressed in rat astrocytes. Next, we determined that OM-LV20 impacted expression changes of TPH1 in CTX-TNA2 cells and exhibited a protective effect on the decrease in cell viability and catalase (CAT) levels induced by hydrogen peroxide. Importantly, we also found that TPH1 expression induced by OM-LV20 may be related to the level of change in the pituitary adenylate cyclase-activating peptide type 1 receptor (PAC1R) and to the JNK signaling pathways, thereby exerting a protective effect on astrocytes against oxidative stress. The protective effects of OM-LV20 likely occur via the ‘PAC1R/JNK/TPH1’ axis, thus highlighting TPH1 as a novel antistroke drug target.

Targeting VIP and PACAP Receptor Signaling: New Insights into Designing Drugs for the PACAP Subfamily of Receptors.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) are neuropeptides involved in a diverse array of physiological and pathological processes through activating the PACAP subfamily of class B1 G protein-coupled receptors (GPCRs): VIP receptor 1 (VPAC1R), VIP receptor 2 (VPAC2R), and PACAP type I receptor (PAC1R). VIP and PACAP share nearly 70% amino acid sequence identity, while their receptors PAC1R, VPAC1R, and VPAC2R share 60% homology in the transmembrane regions of the receptor. PACAP binds with high affinity to all three receptors, while VIP binds with high affinity to VPAC1R and VPAC2R, and has a thousand-fold lower affinity for PAC1R compared to PACAP. Due to the wide distribution of VIP and PACAP receptors in the body, potential therapeutic applications of drugs targeting these receptors, as well as expected undesired side effects, are numerous. Designing selective therapeutics targeting these receptors remains challenging due to their structural similarities. This review discusses recent discoveries on the molecular mechanisms involved in the selectivity and signaling of the PACAP subfamily of receptors, and future considerations for therapeutic targeting.

Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs.

Uterine inflammation is a common pathology in animals, leading to disturbances in reproductive processes and reduced production profitability. Pituitary adenylate cyclase-activating peptide (PACAP) effects at the uterine level during inflammation are not known. In the current study, we analyzed the relative PACAP type 1 receptor (PAC1R) mRNA transcript and protein abundances in the myometrium (MYO), as well s PACAP and PAC1R involvement in the contractile function of inflamed pig uterus. To that end, E. coli suspension (E. coli group) or saline (SAL group) was injected into the uterine horns or laparotomy was performed (CON group). Eight days after the bacteria injections, severe acute endometritis and a reduced relative abundance of PAC1R protein in the MYO were observed. Compared to the period before PACAP in vitro administration, PACAP (10−7 M) in the CON and SAL groups decreased in amplitude in the MYO and endometrium (ENDO)/MYO, whereas in the E. coli group, increased amplitude in the MYO and reduced amplitude in the ENDO/MYO were observed. In the E. coli group, PACAP enhanced the amplitude in the MYO (10−7 M) and decreased the amplitude in the ENDO/MYO (10−8 M) compared with other groups. PACAP (10−7 M) increased the frequency of both kinds of strips in the CON and SAL groups compared with the pretreatment period. PACAP (both doses) did not significantly change the frequency in the E. coli group, whereas in response to PACAP (10−7 M), the frequency was reduced compared to other groups. In the MYO, PAC1R antagonist decreased the amplitude reduction (CON and SAL groups) and reversed a rise in PACAP (10−7 M)-evoked amplitude (E. coli group). PAC1R blocking reversed (MYO) and abolished (ENDO/MYO) the stimulatory effect of PACAP (10−7 M) on the frequency (CON and SAL groups). PAC1R antagonist and PACAP (10−7 M) evoked the appearance of frequency depression in both kinds of strips (E. coli group). In summary, in pigs, severe acute endometritis reduces the relative abundance of PAC1R protein in the MYO, and PAC1R mediates the influence of PACAP on inflamed uterus contractility.

Pharmacologic Characterization of ALD1910, a Potent Humanized Monoclonal Antibody against the Pituitary Adenylate Cyclase-Activating Peptide.

Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

Expression of Pituitary Adenylate Cyclase-activating Peptide, Calcitonin Gene-related Peptide and Headache Targets in the Trigeminal Ganglia of Rats and Humans

Neurotransmitter and headache target localization in the trigeminal ganglia (TG) might increase the understanding of sites of action, and mechanisms related to headache therapy. The overall aim of the study was to investigate the presence of migraine targets in the TG with particular emphasis on pituitary adenylate cyclase-activating peptide (PACAP) and calcitonin gene-related peptide (CGRP), known to be involved in cranial pain processing, and selected headache targets. Rat- and human TG were processed for immunohistochemistry. PACAP-38, CGRP and the headache targets were expressed in rat and human TG. PACAP receptors were confined to neurons and satellite glial cells (SGCs), however with variability between the receptor subtypes PACAP type I receptor (PAC1) and vasoactive intestinal peptide/PACAP receptors 1/2 (VPAC1/2). 5-Hydroxytryptamine (5-HT) receptors were expressed in neuronal somas in rat and human TG (human TG frequency: 5-HT1D > 5-HT1B/1F). Synaptosomal-associated protein 25 kDa (SNAP25) was primarily expressed in SGCs in humans, and neurons in rats, while synaptic vesicle glycoprotein 2A (SV2-A) was confined to SGCs and some neurons in rats and humans. Occasionally, PACAP-38-positive cells also expressed VPAC1, SNAP25 and SV2-A. VPAC1 was generally detected in SGCs enveloping PACAP-38-positive and -negative neuronal somas. Our study revealed potential sites of actions for anti-headache drugs such as PACAP receptor antagonists, Lasmiditan (5-HT1F agonist) and Botox (blocks exocytosis through SV2-A/SNAP25) in rat and human TG and considerable overlap between species in expression to specific cell types, except for VPAC1 and SNAP25.

Hungry Like PACAP Man ‐ The Role of PACAP and PACAP6‐38 in Eating Behaviors

According to the CDC, 34.9% of United States adults are obese, which is linked to premature death, heart disease, cancer, respiratory disorders, fertility problems, Type 2 diabetes, and stroke. Over‐ and under‐eating are related to brain chemistry. A 38 amino acid peptide hormone in the hypothalamus, pituitary adenylate cyclase‐activating peptide (PACAP), may be linked to eating disorders. PACAP binds to PACAP type 1 receptor (PAC1R), a G‐protein coupled receptor. Seven hydrophobic transmembrane (TM) domains hold PAC1R in hypothalamic cell membranes. PAC1R's extracellular domain (ECD) contains a ligand binding site. PAC1R's many negative residues attract PACAP's many positive ECD residues. PACAP's V19, K20, and L27 affect PACAP binding to PAC1R. K20 forms a possible salt bridge with PAC1R's G104, allowing PACAP to align parallel to PAC1R so PACAP's N‐terminus interacts with PAC1R's TM domains. This activates PAC1R, sending a signal inside the cell. Too much PACAP may cause a person to stop eating and lead to eating disorders. PACAP6‐38 is an antagonist formed when a protease removes the first five PACAP residues. When PACAP6‐38 binds to PAC1R, eating increases, possibly leading to obesity. SuJean Choi, PhD wants to determine how ratios of PACAP and PACA6‐38 are regulated. The Greenfield SMART (Students Modeling A Research Topic) Team modeled PAC1R's ECD and its two ligands, PACAP and PACAP6‐38, using 3D printing technology to investigate their relationships. Studying PACAP and PACAP6‐38 regulation and brain chemistry involved in eating behaviors could improve people's lives and decrease obesity‐related US medical costs. Program supported by NIH‐CTSA.

Mice lacking the PACAP type I receptor have impaired photic entrainment and negative masking

The retinohypothalamic tract (RHT) is a retinofugal neuronal pathway which, in mammals, mediates nonimage-forming vision to various areas in the brain involved in circadian timing, masking behavior, and regulation of the pupillary light reflex. The RHT costores the two neurotransmitters glutamate and pituitary adenylate cyclase activating peptide (PACAP), which in a rather complex interplay are mediators of photic adjustment of the circadian system. To further characterize the role of PACAP/PACAP receptor type 1 (PAC1) receptor signaling in light entrainment of the clock and in negative masking behavior, we extended previous studies in mice lacking the PAC1 receptor (PAC1 KO) by examining their phase response to single light pulses using Aschoff type II regime, their ability to entrain to non-24-h light-dark (LD) cycles and large phase shifts of the LD cycle (jet lag), as well as their negative masking response during different light intensities. A prominent finding in PAC1 KO mice was a significantly decreased phase delay of the endogenous rhythm at early night. In accordance, PAC1 KO mice had a reduced ability to entrain to T cycles longer than 26 h and needed more time to reentrain to large phase delays, which was prominent at low light intensities. The data obtained at late night indicated that PACAP/PAC1 receptor signaling is less important during the phase-advancing part of the phase-response curve. Finally, the PAC1 KO mice showed impaired negative masking behavior at low light intensities. Our findings substantiate a role for PACAP/PAC1 receptor signaling in nonimage-forming vision and indicate that the system is particularly important at lower light intensities.

The neuropeptide PACAP upregulates expression and release of cytokines and cell adhesion molecules in human microvascular endothelial cells via VPAC type 1 receptor

Pituitary adenylate cyclase‐activating peptide (PACAP) and vasoactive intestinal peptide (VIP) belong to the same superfamily of neuropeptides which exert their effects by activating G‐protein‐coupled receptors defined as PACAP. So far, three receptor subtypes exist (PAC1R, VPACR‐1 and VPACR‐2). Because, PACAP appears to play a crucial role in cutaneous inflammation and vasoregulation, we examined the expression and biological effects of this peptide in primary human dermal microvascular endothelial cells (HDMECs). We detected the expression of PACAP and VPAC type 1 receptor at RNA and protein level by RT‐PCR and immunohistochemistry, indicating an autocrine regulatory mechanism. cAMP assays revealed VPAC1R to be functional in these cells. RT‐PCR showed upregulation of IL‐8 in a time‐ and dose‐dependent manner. ELISA experiments confirmed release of IL‐8 by HDMEC cells. We also investigated cell adhesion molecule expression after stimulation with PACAP. ICAM‐1 mRNA was upregulated at 3 and 6 h after treatment with PACAP, while VCAM was only upregulated maximally at 6 h after PACAP stimulation, indicating the regulation of cell adhesion molecule expression in human dermal endothelial cells via VPAC1R. Immunoreactivity for VPAC‐1R was enhanced in microvascular endothelial cells of patients with atopic dermatitis and urticaria, indicating upregulation of this receptor in endothelial cells during cutaneous inflammation. In summary, VIP and PACAP may play an important role in cutaneous neurogenic inflammation by activating VPAC‐1R on dermal microvascular endothelial cells.

Phospholipids modulate the biophysical properties and vasoactivity of PACAP-(1--38).

The purpose of this study was to elucidate the interactions between pituitary adenylate cyclase-activating peptide (PACAP)-(1--38) and phospholipids in vitro and to determine whether these phenomena modulate, in part, the vasorelaxant effects of the peptide in the intact peripheral microcirculation. We found that the critical micellar concentration of PACAP-(1--38) was 0.4-0.9 microM. PACAP-(1--38) significantly increased the surface tension of a dipalmitoylphosphatidylcholine monolayer and underwent conformational transition from predominantly random coil in saline to alpha-helix in the presence of distearoyl-phosphatidylethanolamine-polyethylene glycol (molecular mass of 2,000 Da) sterically stabilized phospholipid micelles (SSM) (P < 0.05). Using intravital microscopy, we found that aqueous PACAP-(1--38) evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch that was significantly potentiated when PACAP-(1--38) was associated with SSM (P < 0.05). The vasorelaxant effects of aqueous PACAP-(1--38) were mediated predominantly by PACAP type 1 (PAC(1)) receptors, whereas those of PACAP-(1--38) in SSM predominantly by PACAP/vasoactive intestinal peptide type 1 and 2 (VPAC(1)/VPAC(2)) receptors. Collectively, these data indicate that PACAP-(1--38) self-associates and interacts avidly with phospholipids in vitro and that these phenomena amplify peptide vasoactivity in the intact peripheral microcirculation.

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

Maxadilan is a vasodilatory peptide derived from sand flies that is an agonist at the pituitary adenylate cyclase-activating peptide (PACAP) type 1 receptor. Surprisingly, maxadilan does not share significant sequence homology with PACAP. To examine the relationship between structure and activity of maxadilan, several amino acid substitutions and deletions were made in the peptide. These peptides were examined in vitro for binding to crude membranes derived from rabbit brain, a tissue that expresses PACAP type 1 receptors; and induction of cAMP was determined in PC12 cells, a line that expresses these receptors. The peptides were examined in vivo for their ability to induce erythema in rabbit skin. Substitution of the individual cysteines at positions 1 and 5 or deletion of this ring structure had little effect on activity. Substitution of either cysteine at position 14 or 51 eliminated activity. Deletion of the 19 amino acids between positions 24 and 42 resulted in a peptide with binding, but no functional activity. The capacity of this deletion mutant to interact with COS cells transfected with the PACAP type 1 receptor revealed that this peptide was a specific antagonist to the PACAP type 1 receptor.

The PACAP-type I receptor agonist maxadilan from sand fly saliva protects mice against lethal endotoxemia by a mechanism partially dependent on IL-10.

Sand fly saliva contains maxadilan, a peptide that causes vasodilation and modifies the secretion of pro-inflammatory cytokines by macrophages. We show that 1 to 10 microg maxadilan protected BALB/c mice against a lethal dose of LPS. Maxadilan reduced serum levels of TNF-alpha by approximately tenfold, while it caused a threefold increase in IL-6 and IL-10. The protective effect of maxadilan is partially dependent on its ability to induce IL-10 production since maxadilan did not prevent death from endotoxic shock in IL-10(-/-) mice. Finally, maxadilan is a selective agonist of the pituitary adenylate cyclase-activating peptide (PACAP) type I receptor, and we found that the natural ligand of this receptor (PACAP 38) also protected mice against lethal endotoxemia. These results indicate that activation of the PACAP type I receptor may contribute to the control of systemic inflammation by a mechanism that is partially dependent on IL-10.

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using 35S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [125I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.

The effects of axotomy and preganglionic denervation on the expression of pituitary adenylate cyclase activating peptide (PACAP), galanin and PACAP type 1 receptors in the rat superior cervical ganglion

The effects of axotomy, chemical sympathectomy and preganglionic denervation on the expression of the neuropeptides, pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), and the PACAP type 1 receptor in the rat superior cervical ganglion (SCG) were investigated by immunocytochemistry, in situ hybridization and receptor autoradiography. An antibody recognizing the rat vesicular acetylcholine transporter (VAChT) was used for the detection of preganglionic cholinergic fibers. In the normal SCG, PACAP-immunoreactivity (-IR) was present in numerous, basket-forming, preganglionic nerve fibers, while very few SCG neurons expressed PACAP. GAL-IR was restricted to occasional neurons, and a few nerve fibers, most of which were, in addition, PACAP-IR. PACAP type 1 receptors were expressed in all nerve cell bodies. Axotomy resulted in a rapid and prominent upregulation of PACAP in a large number of nerve cell bodies. There was a large increase also in GAL expression in many nerve cell bodies. In contrast, there was a marked decline in PACAP type 1 receptor expression. Chemical sympathectomy by administration of the catcholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), gave rise to similar changes. Preganglionic denervation led to the disappearance of PACAP- and VAChT-IR baskets and to the upregulation of PACAP and GAL expression in neurons located close to the entrance of the sympathetic chain, whereas PACAP type 1 receptor expression was not affected. PACAP and GAL were coexpressed in most neurons after axotomy and chemical sympathectomy. Taken together, these results indicate that disruption of target contact and/or the infliction of an injury to the axons of the sympathetic neurons, rather than the preganglionic output, regulates the expression of PACAP, GAL and the PACAP type 1 receptor.

Canine adrenal catecholamine response to VIP is blocked by PACAP-(6-27) in vivo.

The goal of the present study was to characterize the adrenal catecholamine response to exogenous vasoactive intestinal peptide (VIP) in anesthetized dogs. We studied the potential involvement of mechanism(s) mediated by muscarinic receptors, L-type Ca2+ channels, VIP-ergic receptors, or pituitary adenylate cyclase-activating peptide (PACAP) receptors. The study consisted of five groups: a vehicle control group receiving VIP (5 micrograms) in the presence of saline and four drug-treated groups receiving VIP (5 micrograms) in the presence of either atropine (500 micrograms), nifedipine (50 micrograms), [Lys1,Pro2,5,Arg3,4,Tyr6]VIP (50 micrograms), or PACAP-(6-27) (50 micrograms). All drugs were locally infused to the left adrenal gland. Plasma catecholamine concentrations were measured in adrenal venous and aortic blood by a high-pressure liquid chromatography-electrochemical method. In the control group, VIP produced a significant increase in adrenal catecholamine output. Neither atropine, nifedipine, nor[Lys1,Pro2,5,Arg3,4,Tyr6]-VIP significantly affected the medullary response to VIP. In the presence of PACAP-(6-27), however, the catecholamine response to VIP was attenuated by approximately 77% (P < 0.05). The present study suggests that adrenal catecholamine secretion induced by exogenous VIP may be mediated by a PACAP-related mechanism, most probably through a PACAP type I receptor, in anesthetized dogs. The data also indicate that neither muscarinic receptors, VIP-ergic receptors, nor dihydropyridine-sensitive L-type Ca2+ channels are operative in the adrenal catecholamine response to exogenous VIP in vivo.

Pituitary adenylate cyclase activating peptide (PACAP) in salivary glands of the rat: origin, and secretory and vascular effects.

Pituitary adenylate cyclase activating peptide (PACAP)-38, injected i.v. to the anaesthetized rat, evoked secretion of saliva from the three major salivary glands, the submandibular glands responding with the greatest and the sublingual glands with the smallest volumes. The parotid saliva was rich in amylase and protein. In vitro, pieces of parotid and submandibular gland tissues released K+ and protein in response to PACAP-38, with atropine and adrenoceptor antagonists present. The blood flow in the submandibular gland increased in response to PACAP-38, despite a marked fall in mean aortic blood pressure. PACAP is a vasoactive intestinal peptide (VIP)-like neuropeptide. A comparison between the two peptides showed PACAP-38 to be more effective than VIP with respect to vascular responses and less or equi-effective with VIP with respect to the secretory responses, thus suggesting the involvement of PACAP type I and type II receptors, respectively PACAP-38 and -27 were present in the parotid gland as judged by radioimmunoassay, the concentration of the former being about twice that of the latter. Parasympathetic denervation, by cutting the auriculo-temporal nerve, reduced the total parotid gland contents of PACAP-38 and -27 by 23 and 44%, respectively (compared with a previously demonstrated 95% reduction of VIP). Sympathetic denervation, section of the facial nerve or treatment with the sensory neurotoxin capsaicin did not affect the content of PACAP. The difference in efficacy between PACAP and VIP in the vascular and secretory responses as well as the difference in localization suggest that the two peptides play different physiological roles in the salivary glands.

PACAP type I receptor activation promotes cerebellar neuron survival through the cAMP/PKA signaling pathway.

The molecular nature, transduction pathways, and neurotrophic functions of pituitary adenylate cyclase activating peptide (PACAP) receptors were studied in primary culture of rat cerebellar granule cells. We show that cerebellar neurons express several PACAP type I receptor (PVR I) isoforms, including the short (PVR Is) and the Hop (PVR I-Hop) splice variants, the latter being restricted to neurons and not found in cerebellar glial cell cultures. In vitro, cerebellar granule cells die rapidly in the absence of a high concentration of K+ (25 mM), as demonstrated by TUNEL histochemistry, which shows that K+ deprivation induces massive neuronal apoptosis within 12 hr. This effect was reversed by PACAP 27 and 38. Both forms of PACAP prevent DNA fragmentation and allow long-term neuronal survival in the absence of high K+ (as shown by MAP2 immunostaining) and stimulate a reporter gene driven by the full-length c-fos promoter. These effects of PACAP are fully abolished upon transient transfection of cells with a dominant inhibitory mutant of the cAMP-dependent protein kinase (PKA). Taken together, these results show that in cerebellar granule neurons, PACAP type I receptors regulate gene expression and promote neuronal survival through the cAMP/PKA pathway.

PACAP induces bradycardia in guinea-pig heart by stimulation of atrial cholinergic neurones.

Based on previous studies which indicated that pituitary adenylate cyclase activating peptide (PACAP) acts as a positive inotropic and chronotropic substance in different species via the cAMP signal transduction pathway, the objective of the present work was to investigate cAMP-regulated myocardial key proteins in response to PACAP in isolated ventricular cells of the guinea pig. Surprisingly, the two molecular forms of PACAP, PACAP(1-27) and PACAP(1-38), showed no effect on intracellular cAMP-levels, L-type Ca2+ channel current or phosphorylation of troponin inhibitor (TnI) and phospholamban (PLB). Additionally, inotropy of isolated guinea-pig ventricular strips was not affected by the neuropeptide. However, in isolated spontaneously beating guinea-pig atria, PACAP(1-27) and PACAP(1-38), but not VIP induced severe bradycardia in a dose-dependent manner. This effect could be prevented by preincubation with the PACAP receptor antagonist PACAP(6-38), by atropine and by omega-conotoxin, a blocker of neuronal N-type Ca2+ channels. PACAP stimulates release of [3H]-labelled acetylcholine. Only preparations showing an increase in [3H]acetylcholine release developed bradycardia, indicating a causal relationship between both phenomena. It was concluded that PACAP exerts no influence on guinea-pig ventricular tissue, but induces negative chronotropic effects in isolated guinea-pig atria by stimulation of acetylcholine release from parasympathetic neurons via PACAP type 1 receptors.

Expression of pituitary adenylate cyclase activating peptide (PACAP) and PACAP type I receptors in the rat adrenal medulla

Expression of pituitary adenylate cyclase activating peptide (PACAP) and PACAP type I receptors in the rat adrenal medulla

Expression of pituitary adenylate cyclase activating peptide (PACAP) and PACAP type I receptors in the rat adrenal medulla

Pituitary adenylate cyclase activating polypeptide (PACAP) which belongs to the vasoactive intestinal polypeptide family of regulatory peptides, occurs in two variants, PACAP-27 and PACAP-38, and is thought to be an important messenger in both the central and peripheral nervous system. Three cloned G-protein coupled 7 transmembrane spanning receptors bind PACAP with high affinity, one reacting only weakly with VIP (PACAP type I receptor) the other two binding vasoactive intestinal peptide (VIP) with equally high affinity (VIP type I and II receptors). The PACAP type I receptor displays high affinity for both variants of PACAP. In this study, we have investigated the distribution of PACAP and the PACAP type I receptor in the adrenal medulla of newborn and adult rat. Immunocytochemistry revealed, in the adult rat, a dense network of PACAP-immunoreactive nerve fibers terminating on chromaffin cells. Such fibers were few and weakly immunoreactive in the newborn rat. PACAP-immunoreactive medullary cells could not be detected in the adult rat, whereas in the newborn, occasional cells were seen. By in situ-hybridization we detected PACAP type I receptor mRNA in a majority of the adrenal medullary cells of both newborn and adult rat. Receptor autoradiography using 125I-PACAP-27 as ligand revealed binding-sites with a localization virtually identical to the in situ hybridization signal indicating a functional expression of high-affinity type 1 PACAP receptors in the adrenal medulla. Additionally, in the adult rat, single or clustered large cells, presumably ganglion cells, contained an even higher abundance of PACAP receptor mRNA as well as binding sites than the surrounding chromaffin cells. Our observations on the distribution of PACAP peptide and PACAP receptors in the adrenal medulla suggest that both chromaffin cells and ganglion cells are PACAP targets. The data thus strengthen earlier observations indicating an important regulatory role of PACAP in catecholamine biosynthesis and release. The presence of both ligand and receptors in newborn rats may indicate a role for PACAP in the development of the adrenal medulla.