David Vaudry

Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally α-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications.

Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide: IUPHAR Review 1

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are members of a superfamily of structurally related peptide hormones that includes glucagon, glucagon‐like peptides, secretin, gastric inhibitory peptide (GIP) and growth hormone‐releasing hormone (GHRH). VIP and PACAP exert their actions through three GPCRs – PAC1, VPAC1 and VPAC2– belonging to class B (also referred to as class II, or secretin receptor‐like GPCRs). This family comprises receptors for all peptides structurally related to VIP and PACAP, and also receptors for parathyroid hormone, corticotropin‐releasing factor, calcitonin and related peptides. PAC1 receptors are selective for PACAP, whereas VPAC1 and VPAC2 respond to both VIP and PACAP with high affinity. VIP and PACAP play diverse and important roles in the CNS, with functions in the control of circadian rhythms, learning and memory, anxiety and responses to stress and brain injury. Recent genetic studies also implicate the VPAC2 receptor in susceptibility to schizophrenia and the PAC1 receptor in post‐traumatic stress disorder. In the periphery, VIP and PACAP play important roles in the control of immunity and inflammation, the control of pancreatic insulin secretion, the release of catecholamines from the adrenal medulla and as co‐transmitters in autonomic and sensory neurons. This article, written by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC‐IUPHAR) subcommittee on receptors for VIP and PACAP, confirms the existing nomenclature for these receptors and reviews our current understanding of their structure, pharmacology and functions and their likely physiological roles in health and disease. More detailed information has been incorporated into newly revised pages in the IUPHAR database (http://www.iuphar‐db.org/DATABASE/FamilyMenuForward?familyId=67).

PACAP protects cerebellar granule neurons against oxidative stress-induced apoptosis

Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in neuronal cell death associated with neurodegenerative diseases and stroke. In the present study, we have investigated the potential neuroprotective effect of pituitary adenylate cyclase‐activating polypeptide (PACAP) on oxidative stress‐induced apoptosis. Incubation of cerebellar granule cells with PACAP inhibited hydrogen peroxide‐evoked cell death in a concentration‐dependent manner. The effect of PACAP on granule cell survival was not mimicked by vasoactive intestinal polypeptide and was blocked by the antagonist PACAP6‐38. The protective action of PACAP upon hydrogen peroxide‐induced neuronal cell death was abolished by the MAP‐kinase kinase (MEK) inhibitor U0126 and mimicked by the caspase‐3 inhibitor Z‐DEVD‐FMK. PACAP markedly inhibited hydrogen peroxide‐evoked caspase‐3 activation and DNA fragmentation. Taken together, these data indicate that PACAP, acting through PACAP receptor type 1, exerts a potent protective effect against neuronal degeneration induced by hydrogen peroxide. The anti‐apoptotic effect of PACAP is mediated through the MAP‐kinase pathway and can be accounted for by inhibition of caspase‐3 activation resulting from oxidative stress.

Pituitary adenylate cyclase-activating polypeptide promotes cell survival and neurite outgrowth in rat cerebellar neuroblasts.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors have been detected in the rat cerebellum during ontogenesis. In particular, PACAP receptors are actively expressed in immature granule cells, suggesting that PACAP may act as a neurotrophic factor in the developing rat cerebellum. In the present study, we have investigated the effect of PACAP on cell survival and neurite outgrowth in cultured immature cerebellar granule cells. In control conditions, cultured granule cells undergo programmed cell death. Exposure of cultured cells to PACAP for 24 and 48 h provoked a significant increase in the number of living cells. The effect of PACAP on cell survival was inhibited by the PACAP antagonist PACAP(6-38). Vasoactive intestinal polypeptide was approximately 1000 times less potent than PACAP in promoting cell survival. PACAP also induced a significant increase in the number of processes and in the cumulated length of neurites borne by cultured neuroblasts. The present results demonstrate that PACAP promotes cell survival and neurite outgrowth in cultured immature granule cells. Since PACAP and its receptors are expressed in situ in the rat cerebellar cortex, these data strongly suggest that PACAP plays a physiological role in the survival and differentiation of cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.

Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal

Significance Inflammation often complicates diseases associated with oxidative stress. This study shows that inflammatory macrophages release proteins with specific forms of cysteine oxidation to disulfides, particularly glutathionylation. Redox proteomics identified peroxiredoxin 2 (PRDX2) as a protein released in glutathionylated form by inflammation both in vivo and in vitro. Extracellular PRDX2 then triggers the production of TNF-α. These data indicate that redox-dependent mechanisms, in an oxidative cascade, can induce inflammation. The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the –SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well‐established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.

High Expression of Transient Receptor Potential Channels in Human Breast Cancer Epithelial Cells and Tissues: Correlation with Pathological Parameters

Background: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. Methods: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. Results: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. Conclusion: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.

Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase‐activating polypeptide (PACAP) prevents C2‐ceramide‐induced apoptosis. The neuroprotective effect of PACAP was dose‐dependent and blocked by its antagonist, PACAP6‐38, whereas the PACAP‐related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl‐cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl‐cyclase and MAP‐kinase pathways contribute to the neuroprotective action of the peptide. C2‐ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP‐kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA‐ and PKC‐independent mechanism. C2‐ceramide induced a time‐dependent activation of caspase‐3, enhanced the amount of cleaved caspase‐3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2‐ceramide‐induced activation of caspase‐3, reduced the expression of cleaved caspase‐3 and blocked DNA fragmentation. Taken together, the present results show that C2‐ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase‐3. Our data also demonstrate that PACAP is a potent inhibitor of C2‐ceramide‐induced apoptosis.

Comparative Distribution of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Binding Sites and PACAP Receptor mRNAs in the Rat Brain During Development

The distribution and density of pituitary adenylate cyclase‐activating polypeptide (PACAP) binding sites as well as PACAP‐specific receptor 1 (PAC1‐R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1‐R), and VPAC2‐R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27‐amino‐acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3‐ to 5‐fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [35S] uridine triphosphate‐specific riboprobes. The evolution of the distribution of PAC1‐R and VPAC1‐R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1‐R mRNA being much lower than that of PAC1‐R mRNA. In contrast, intense expression of VPAC2‐R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1‐R and VPAC1‐R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1‐R and/or VPAC1‐R, may play a crucial role in the regulation of neurogenesis in the rat brain. J. Comp. Neurol. 425:495–509, 2000.