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.     

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.     

Role of VIP and PACAP in islet function

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two closely related neuropeptides that are expressed in islets and in islet parasympathetic nerves. Both peptides bind to their common G-protein-coupled receptors, VPAC1 and VPAC2, and PACAP, in addition to the specific receptor PAC1, all three of which are expressed in islets. VIP and PACAP stimulate insulin secretion in a glucose-dependent manner and they both also stimulate glucagon secretion. This action is achieved through increased formation of cAMP after activation of adenylate cyclase and stimulation of extracellular calcium uptake. Deletion of PAC1 receptors or VPAC2 receptors results in glucose intolerance. These peptides may be of importance in mediating prandial insulin secretion and the glucagon response to hypoglycemia. Animal studies have also suggested that activation of the receptors, in particular VPAC2 receptors, may be used as a therapeutic approach for the treatment of type 2 diabetes. This review summarizes the current knowledge of the potential role of VIP and PACAP in islet function.     

Immunohistochemical localization and distribution of VIP/PACAP receptors in human lung

VIP and PACAP are distributed in nerve fibers throughout the respiratory tract acting as potent bronchodilators and secretory agents. By using RT-PCR and immunoblotting techniques, we have previously shown the expression of common VIP/PACAP (VPAC(1) and VPAC(2)) and specific PACAP (PAC(1)) receptors in human lung. Here we extend our aims to investigate by immunohistochemistry their localization and distribution at this level. A clear immunopositive reaction was obtained in human lung sections by using either anti-VPAC(1) or -VPAC(2) receptor antibodies but not with anti-PAC(1) receptor antibody. However, PAC(1) receptor (and VPAC(1) and VPAC(2) receptors) could be identified in lung membranes by immunoblotting which supports that the PAC(1) receptor is expressed at a low density. Both VPAC(1) and VPAC(2) receptors showed similar immunohistochemical patterns appearing in smooth muscle cells in the wall of blood vessels and in white blood cells (mainly in areas with inflammatory responses). The results agree with previous evidence on the importance of both peptides in the immune system and support their anti-inflammatory and protective roles in lung.     

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.     

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.     

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.     

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.     

Neuroleptic Drugs and PACAP Differentially Affect the mRNA Expression of Genes Encoding PAC1/VPAC Type Receptors

Several lines of evidence suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide playing an important role as a neuromodulator. It has been indicated that PACAP is associated with mental diseases, and that regulation of the PACAPergic signals could be a potential target for the treatment of such psychiatric states as schizophrenia. Recent studies have suggested that action of neuroleptic drugs is mediated not only by dopaminergic and serotonergic neurotransmission, but also via neuropeptides which may act both as neurotransmitters and as neuromodulators. The present study examines whether currently-used neuroleptics influence the action of PACAP receptors, whose expression is altered in a schizophrenic patient. Real-time polymerase chain reaction (PCR) was used to examine the effects of haloperidol, olanzapine and amisulpride on the expression of genes coding PAC1/VPAC type receptors in the T98G glioblastoma cell line, as an example of an in vitro model of glial cells. PAC1 mRNA expression fell after 24-h incubation with haloperidol or olanzapine; however the effect was not maintained after 72 h, and haloperidol even up-regulated PAC1 mRNA expression in a dose-dependent manner. All the examined drugs decreased VPAC2 mRNA expression, especially after 72-h incubation. Haloperidol (typical neuroleptic) was distinctly more potent than atypical neuroleptic drugs (olanzapine and amisulpride). In addition, PACAP increased PAC1 and VPAC2 mRNA expression. In conclusion, our findings suggest PACAP receptors may be involved in the mechanism of typical and atypical neuroleptic drugs.     

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.     

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.     

Molecular pharmacology and structure of VPAC Receptors for VIP and PACAP

VIP and PACAP are two prominent neuropeptides which share two common G protein-coupled receptors VPAC1 and VPAC2 while PACAP has an additional specific receptor PAC1. This paper reviews the present knowledge regarding three aspects of VPAC receptors including: (i). receptor specificity towards natural VIP-related peptides and pharmacology of synthetic agonists or antagonists; (ii). receptor signaling; (iii). molecular basis of ligand-receptor interaction as determined by site-directed mutagenesis, construction of receptor chimeras and structural modeling.     

VIP and PACAP analogs regulate therapeutic targets in high-risk neuroblastoma cells

Neuroblastoma (NB) is a pediatric cancer. New therapies for high-risk NB aim to induce cell differentiation and to inhibit MYCN and ALK signaling in NB. The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are 2 related neuropeptides sharing common receptors. The level of VIP increases with NB differentiation. Here, the effects of VIP and PACAP analogs developed for therapeutic use were studied in MYCN-amplified NB SK-N-DZ and IMR-32 cells and in Kelly cells that in addition present the F1174L ALK mutation. As previously reported by our group in IMR-32 cells, VIP induced neuritogenesis in SK-N-DZ and Kelly cells and reduced MYCN expression in Kelly but not in SK-N-DZ cells. VIP decreased AKT activity in the ALK-mutated Kelly cells. These effects were PKA-dependent. IMR-32, SK-NDZ and Kelly cells expressed the genes encoding the 3 subtypes of VIP and PACAP receptors, VPAC1, VPAC2 and PAC1. In parallel to its effect on MYCN expression, VIP inhibited invasion in IMR-32 and Kelly cells. Among the 3 PACAP analogs tested, [Hyp²]PACAP-27 showed higher efficiency than VIP in Kelly cells. These results indicate that VIP and PACAP analogs act on molecular and cellular processes that could reduce aggressiveness of high-risk NB.     

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.