PAC1 Receptor

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Autocrine Expression and Ontogenetic Functions of the PACAP Ligand/Receptor System during Sympathetic Development

In recent years, it has become evident that neural stem cells in the adult mammalian brain continuously generate new neurons, mainly in the hippocampus and olfactory bulb. Although different growth factors have been shown to stimulate neurogenesis in the adult brain, very little is known about the role of neuropeptides in this process. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic effects acting through three receptors to which it has high affinity, namely, PACAP receptor 1 (PAC1), vasoactive intestinal peptide (VIP) receptor 1, and VIP receptor 2. We show that PAC1 is expressed in the neurogenic regions of the adult mouse brain, namely the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus. Cultured neural stem cells isolated from the lateral ventricle wall of adult mice express PAC1 and proliferate in vitro in response to two PAC1 agonists, PACAP and Maxadilan, but not VIP at physiologic concentrations, indicating PAC1 as a mediator of neural stem cell proliferation. Pharmacologic and biochemical characterization of PACAP-induced neural stem cell proliferation revealed the protein kinase C pathway as the principal signaling pathway, whereas addition of epidermal growth factor synergistically enhanced the proliferating effect of PACAP. Further in vitro characterization of the effect of PACAP on neural stem cells showed PACAP capable of stimulating ex novo in vitro formation of multipotent neurospheres with the capacity to generate both neuronal and glial cells. Finally, intracerebroventricular infusion of PACAP increases cell proliferation in the ventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus. We conclude that PACAP, through PAC1, is a potent mediator of adult neural stem cell proliferation.

PACAP-PAC1 receptor inhibition is effective in opioid induced hyperalgesia and medication overuse headache models

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide that produces its biological effects by interacting with G protein-coupled receptors. Molecular cloning of the PACAP receptor revealed the existence of five splice variant receptor forms differing in the third intracellular loop region, with four variants activating both adenylyl cyclase and phosphoinositide phospholipase C and one variant activating only adenylyl cyclase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report cloning of a novel PACAP receptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously cloned short form of the PACAP receptor (PACAPR) primarily by discrete sequences located in transmembrane domains II and IV. Reverse transcriptase-polymerase chain reaction and primer extension analyses demonstrated tissue-specific differential expression of mRNAs encoding PACAPR TM4 and splice variant forms of the PACAP receptor. PACAPR TM4 and PACAPR possess identical intracellular domains, implicated as primary determinants of G protein recognition by rhodopsin-like receptors. However, unlike the PACAPR, PACAPR TM4 does not activate either adenylyl cyclase or phosphoinositide phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates increases in [Ca2+]i in cells expressing PACAPR TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with vasoactive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is characteristic of the PACAP receptor involved in regulation of insulin secretion from pancreatic beta islets, a tissue expressing transcripts for PACAPR TM4 but not for PACAPR or its longer splice variant forms. These findings are consistent with a role of PACAPR TM4 in the physiological control of insulin release by PACAP in beta-islet cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intracellular domains identical to those of the PACAPR, suggests that receptor-G protein recognition by rhodopsin-like receptors can be determined by sequences other than those located in intracellular receptor domains.

Region-specific changes in the immunoreactivity of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC2, and PAC1 receptor) in the aged rat brains

Pituitary adenylate cyclase‐activating peptide (PACAP) is a neuropeptide which exists in two biologically active forms; PACAP‐27 and PACAP‐38. PACAP is widely expressed in the brain and periphery where it is involved in many physiological processes including pain, inflammation, stress and anxiety. PACAP potently activates three different G protein‐coupled receptors (GPCRs); the PAC1 receptor, the VPAC1 receptor and VPAC2 receptor. The VPAC receptors are co‐receptors for PACAP and vasoactive intestinal peptide (VIP). The PAC1 receptor has several known splice variants in the N‐terminal domain, which include the PAC1n receptor (full N‐terminus) and the PAC1s receptor (21 amino acid N‐terminal deletion). To develop therapeutics targeting the PACAP system, it is essential to understand how these receptors signal and how this signalling is inhibited. Here we pharmacologically characterised the signalling capabilities of PACAP‐responsive receptors and how these responses were blocked by antagonists.The pharmacology of the human PAC1n, PAC1s, VPAC1, VPAC2 receptors were examined in transfected Cos7 cells. Using concentration‐response curves, the ability of PACAP and related peptides to stimulate cAMP, IP1, pAkt, pERK and pCREB responses were measured. Signalling bias was then quantified. The ability of multiple antagonists to block PACAP‐38, PACAP‐27 or VIP‐induced cAMP responses at PACAP‐responsive receptors was then determined.PACAP‐responsive receptors stimulated activation of all signalling molecules measured. However, only limited signalling bias was observed. For example, VIP was 4‐fold biased towards cAMP production over IP1 production at the PAC1n receptor. Interestingly, the PAC1n and PAC1s receptors displayed distinct pharmacology. At the PAC1s receptor, VIP was found to be 20‐fold more potent than at the PAC1n receptor. Probe‐ or agonist‐dependent antagonism was observed at the PAC1n and PAC1s receptors. Both VIP and PACAP‐27 were more potently antagonised than PACAP‐38 by the antagonists PACAP6–38 and M65.The distinct pharmacological profile displayed by the PAC1s receptor, suggests that it can act as a dual receptor for VIP and PACAP under specific conditions. Furthermore, differences in antagonist pharmacology at PACAP‐responsive receptors suggests the effectiveness of blocking a signalling pathway can be influenced by which endogenous agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PAC1 receptor.Support or Funding InformationThis research is supported by grants from the Royal Society of New Zealand Marsden Fund (RSNZ) and the Health Research Council of New Zealand (HRC).

Cloning and expression of a complementary DNA encoding the bovine receptor for pituitary adenylate cyclase-activating polypeptide (PACAP)

This study aimed to provide proof-of-concept that multi-receptor antagonist peptides can be generated by covalently linking independent antagonist peptides that block calcitonin gene-related peptide (CGRP8-37) or pituitary adenylate cyclase-activating peptide (PACAP)/vasoactive intestinal peptide (VIP) (PACAP6-38) activity. The neuropeptides CGRP and PACAP are implicated in migraine and pain pathogenesis. CGRP and PACAP are elevated during a migraine attack, and following infusion of either peptide, patients develop migraine-like attacks. This indicates that targeting both these systems may provide therapeutic benefits. Mechanistic studies suggest that these peptides largely act independently from one another. Therefore, blocking the activity of both CGRP and PACAP simultaneously could provide a clinical advantage over individual blockade. One strategy is to develop a single antagonist capable of inhibiting the signaling of both CGRP and PACAP receptors, a multi-receptor antagonist. N-terminal truncation of CGRP and PACAP generates the antagonists CGRP8-37 and PACAP6-38, respectively. These are commonly used as research tools for the CGRP and PACAP receptors. These peptide antagonists were, therefore, used as the basis for the design of multi-receptor antagonists against the CGRP and PACAP receptors and to test their functionality invitro. To generate multi-receptor antagonists, CGRP8-37 was linked through 1,3-dipolar cycloaddition using click chemistry to PACAP6-38 at amino acid residues 21, 34, or 38. The ability of these multi-receptor antagonists to block CGRP activity (CGRP and AMY1 receptors) and PACAP-38, PACAP-27, and VIP activity (PAC1, VPAC1, and VPAC2 receptors) was then characterized in transfected Cos7 cells. The peptides were then further examined in pain-relevant rat spinal cord cultures, as a model of endogenous receptors. Multi-receptor antagonists were successfully generated, displaying similar antagonist potency to their parental antagonists in both transfected Cos7 cells and in spinal cord cultures. Interestingly, CGRP8-37 linked to position 38 of PACAP6-38 was a more potent antagonist of CGRP activity than CGRP8-37. This study provides proof-of-concept evidence for the development of potent multi-receptor antagonists capable of blocking both CGRP and PACAP activity.

Signaling pathways in PACAP regulation of VIP gene expression in human neuroblastoma cells

The control of saliva secretion is mainly under the autonomic nervous control. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is now recognized as the multi‐functional neuropeptide in various organs. Vasoactive intestinal polypeptide (VIP) is similar function to PACAP. In this study, we compared the distribution of PACAP receptor, PAC1R and VIP receptor, VPAC1, in major salivary glands; parotid, submandibular and sublingual glands, and examined the effect of PACAP on saliva secretion in 8 weeks old C57BL/6 mice. In parotid gland, PAC1R and VPAC1 were detected in the cells of striated duct. In the case of submandibular gland, PAC1R and VPAC1 were detected in the tall columnar epithelial cells, called pillar cells, in granular ducts and some of the cells in the striated ducts. In sublingual gland, PAC1R and VPAC1 were expressed mainly in the epithelial cells of striated ducts. Intranasal injection of PACAP enhanced the secretion of saliva. Taken together, these results indicated that the distribution of PAC1R and VPAC1 were similar in three major salivary glands and that PACAP and VIP might be the useful agents for the future dry mouth therapuutics.

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel peptide that was isolated from ovine hypothalamic tissue on the basis of its ability to stimulate cAMP accumulation in cultured rat pituitary cells. Recently we demonstrated that PACAP can stimulate cAMP accumulation and secretory function in cultured rat Sertoli cells. Since ovarian granulosa cells share many properties with Sertoli cells, we have examined the effect of PACAP (consisting of 38 or 27 amino acid residues) on cultured granulosa cell function. Granulosa cells were obtained from the ovaries of 25-day-old rats implanted with a silastic capsule containing diethylstilbestrol 5 days prior to culture. PACAP 38 (0.1 microM-0.01 pM), both alone and in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine, stimulated cAMP accumulation 4-8-fold with an ED50 of approximately 100 pM. Maximal PACAP 38 or PACAP 27 stimulation of granulosa cell cAMP was significantly greater than that produced by a maximally effective concentration of FSH. Because PACAP 38 and 27 have 68% sequence homology with vasoactive intestinal peptide (VIP), and since VIP stimulates granulosa cell cAMP accumulation and estradiol and progesterone secretion, we examined the possibility that PACAP could be acting via the VIP receptor. VIP stimulated cAMP only at concentrations of 10 nM or greater, whereas the PACAP stimulation was evident at 10 pM. Moreover, only one of three potent VIP antagonists inhibited VIP stimulation of cAMP accumulation, and only at 1 microM or greater. This VIP antagonist did not inhibit PACAP 38 action at 2000-fold excess concentration. Interestingly PACAP 38 was more effective than PACAP 27 with regard to steroid secretion and the ability to induce LH responsiveness. PACAP and VIP stimulation of granulosa cell cAMP accumulation or estradiol or progesterone secretion was not additive. Thus, these data support the hypothesis that granulosa cells have specific PACAP 38 receptors and that VIP acts via these receptors. In addition, PACAPs 38 and 27 are more potent stimulators of cAMP accumulation in luteinized granulosa cells than LH. These results both pre- and postovulation, along with previous data indicating that the PACAPs are found in the ovaries, suggest a role for PACAP in the regulation of ovarian function.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC1 and VPAC2, in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC1 and VPAC2 receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC2 receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC1 receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses. J. Neurosci. Res. 57:953–961, 1999. © 1999 Wiley-Liss, Inc.

Pituitary adenylate cyclase activating polypeptide (PACAP) inhibited dose dependently the DNA synthesis stimulated by arginine vasopressin (AVP) in cultured rat aortic smooth muscle cells (SMC). The inhibition was cell cycle dependent and the maximum inhibition was observed when added at the late G1 phase of the cell cycle. Vasoactive intestinal polypeptide (VIP), which shows a considerable homology with PACAP, also inhibited dose dependently the AVP-induced DNA synthesis in a cell cycle dependent manner. The maximum inhibition was also observed at the late G1 phase. The patterns of both the dose-dependent inhibitions were similar, and the inhibition by a combination of PACAP and VIP was not additive. PACAP stimulated dose dependently cAMP accumulation in aortic SMC. VIP also stimulated cAMP accumulation, and the accumulation by a combination of PACAP and VIP was not additive. Both PACAP and VIP had little effect on phosphoinositide hydrolysis in these cells. The suppression of the AVP-induced DNA synthesis by PACAP or VIP was enhanced by 3-isobutyl-1-methylxanthine, an inhibitor for phosphodiesterases. Dibutyryl cAMP, but not 8-bromo-cGMP, inhibited the AVP-induced DNA synthesis, and a combination of PACAP and dibutyryl cAMP was not additive. [Ac-Tyr1,D-Phe2]growth hormone-releasing factor, an antagonist for VIP receptor, reversed the inhibitory effect of PACAP on the AVP-induced DNA synthesis. These results suggest that PACAP has an antiproliferative effect on aortic SMC at the late G1 phase of the cell cycle through cAMP production, and that PACAP and VIP inhibit the AVP-induced DNA synthesis by a common mechanism.