Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.     

A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists

Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.     

Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

Role of PACAP and VIP in astroglial functions

Astrocytes represent at least 50% of the volume of the human brain. Besides their roles in various supportive functions, astrocytes are involved in the regulation of stem cell proliferation, synaptic plasticity and neuroprotection. Astrocytes also influence neuronal physiology by responding to neurotransmitters and neuropeptides and by releasing regulatory factors termed gliotransmitters. In particular, astrocytes express the PACAP-specific receptor PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R during development and/or in the adult. There is now clear evidence that PACAP and VIP modulate a number of astrocyte activities such as proliferation, plasticity, glycogen production, and biosynthesis of neurotrophic factors and gliotransmitters.     

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

GI side-effects of a possible therapeutic GRF analogue in monkeys are likely due to VIP receptor agonist activity.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.     

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.     

VIP and PACAP stimulate TSH release from the bullfrog pituitary

We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin (TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal polypeptide (VIP) (fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24h with graded concentrations of fVIP, fPACAP38 and PACAP27 (10(-9) to 10(-6)M) induced a dose-dependent stimulation of TSH release with minimum effective doses of 10(-9)M for fVIP and 10(-8)M for fPACAP38 and PACAP27. The PAC1-R/VPAC2-R antagonist PACAP(6-38) (10(-7) and 10(-6)M) dose-dependently suppressed the stimulatory effects of fVIP and fPACAP38 (10(-7)M each). Likewise, this antagonist (10(-6) and 10(-5)M) dose-dependently attenuated the stimulatory effect of PACAP27 (10(-7)M). On the other hand, the VPAC1-R/VPAC2-R antagonist [d-pCl-Phe(6), Leu(17)]VIP (10(-6) and 10(-5)M) dose-dependently inhibited the stimulatory effect of fVIP (10(-9)M) and PACAP27 (10(-8)M), but did not affect the response to fPACAP38 (10(-8)M). These data indicate that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R.     

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.     

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用

垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide,PACAP）特异受体PAC1-R (PACAP receptor 1)是神经系统疾病药物开发的重要靶点。为了研究其表达的生物学机制,本研究克隆了PAC1-R基因转录起始位点上游从-2 500到+26的2 526 bp启动子片段,构建PAC1-R启动子驱动的荧光素酶基因报告载体pGL3-PAC1-Rp,并确证PAC1-R启动子荧光素酶报告系统在小鼠脑神经瘤细胞Neuro-2a和人神经母细胞瘤细胞SH-SY5Y中工作正常。运用此PAC1-R启动子荧光素酶报告系统,首次发现低浓度过氧化氢（hydrogen peroxide,H2O2）有效激活PAC1-R启动子,此作用可被转录因子特化蛋白1（specificity protein 1,SP1）抑制剂光神霉素A（mithramycin A）所抑制,提示SP1参与介导H2O2对PAC1-R启动子的激活作用。生物信息学分析显示,PAC1-R启动子含有多个SP1结合位点。PAC1-R启动子的荧光素酶报告系统的构建为深入探索PAC1-R高表达的作用与机制奠定了基础,低浓度H2O2对PAC1-R启动子激活作用的发现有助于深入诠释低浓度活性氧的生理学作用。     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.     

PAC1 receptors mediate pituitary adenylate cyclase-activating polypeptide- and progesterone-facilitated receptivity in female rats

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a feed-forward, paracrine/autocrine factor in the hypothalamic ventromedial nucleus (VMN) for receptivity and sensitizes pituitary hormone release for ovulation. The present study examined receptor(s) and signaling pathway by which PACAP enhances rodent lordosis. PACAP binds to PACAP (PAC1)- and vasoactive intestinal peptide-preferring receptors (VPAC1, VPAC2). Ovariectomized rodents primed with estradiol (EB) were given PACAP or vasoactive intestinal peptide directly onto VMN cells. Only PACAP facilitated receptivity. Pretreatment with VPAC1 and VPAC2 inhibitors blocked both PACAP- and progesterone (P)-induced receptivity. Antisense (AS) oligonucleotides to PAC1 (not VPAC1 or VPAC2) inhibited the behavioral effect of PACAP and P. By real-time RT-PCR, EB, P and EB+P enhanced VMN mRNA expression of PAC1. Within the total PAC1 population, EB and EB+P induced expression of short form PAC1 and PAC1hop2 splice variants. Finally, blocking cAMP/protein kinase A signaling cascade by antagonists to cAMP activity and protein kinase A or by antisense to dopamine- and cAMP-regulated phosphoprotein of 32 kDa blocked the PACAP effect on behavior. Collectively, these findings provide evidence that progesterone receptor-dependent receptivity is, in part, dependent on PAC1 receptors for intracellular VMN signaling and delineate a novel, steroid-dependent mechanism for a feed-forward reinforcement of steroid receptor-dependent reproductive receptivity.     

Neurotrophic effects of PACAP in the cerebellar cortex

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.