Targeting VIP and PACAP receptor signalling: new therapeutic strategies in multiple sclerosis

MS (multiple sclerosis) is a chronic autoimmune and neurodegenerative pathology of the CNS (central nervous system) affecting approx. 2.5 million people worldwide. Current and emerging DMDs (disease-modifying drugs) predominantly target the immune system. These therapeutic agents slow progression and reduce severity at early stages of MS, but show little activity on the neurodegenerative component of the disease. As the latter determines permanent disability, there is a critical need to pursue alternative modalities. VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating peptide) have potent anti-inflammatory and neuroprotective actions, and have shown significant activity in animal inflammatory disease models including the EAE (experimental autoimmune encephalomyelitis) MS model. Thus, their receptors have become candidate targets for inflammatory diseases. Here, we will discuss the immunomodulatory and neuroprotective actions of VIP and PACAP and their signalling pathways, and then extensively review the structure–activity relationship data and biophysical interaction studies of these peptides with their cognate receptors.     

Anorexigenic effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide in the chick brain are mediated by corticotrophin-releasing factor

Intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide-38 (PACAP) or vasoactive intestinal peptide (VIP) inhibits feeding in chicks. However, the underlying anorexigenic mechanism(s) has not yet been investigated. The present study investigated whether these peptides influence the activity of corticotrophin-releasing factor (CRF) neural pathways in the brain of chicks. Firstly, we found that ICV injections of PACAP and VIP increased plasma corticosterone concentrations. The corticosterone-releasing effect of PACAP was completely attenuated by co-injection of astressin, a CRF receptor antagonist, but this effect was only partial for VIP. These results demonstrated that CRF neurons mediate the actions of PACAP and, to a lesser extent, VIP, and suggest that the signaling mechanisms differ between the two peptides. This difference may arise from the two peptides interacting with different receptors because the corticosterone-releasing effect of PACAP, but not VIP, was completely attenuated by co-injection of PACAP (6–38), a PACAP receptor antagonist. Finally, we examined the effect of ICV co-injection of astressin on the anorexigenic effects of PACAP and VIP and found that the effects of both peptides were attenuated by astressin. Overall, the present study suggests that the anorexigenic effects of PACAP and VIP are mediated by the activation of CRF neurons.     

Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor

Atrial natriuretic peptide (ANP) and the closely-related peptides BNP and CNP are highly conserved cardiovascular hormones. They bind to single transmembrane-spanning receptors, triggering receptor-intrinsic guanylyl cyclase activity. The "truncated" type-C natriuretic peptide receptor (NPR-C) has long been called a clearance receptor because it lacks the intracellular guanylyl cyclase domain, though data suggest it might negatively couple to adenylyl cyclase via G(i). Here we report the molecular cloning and characterization of the Xenopus laevis type-C natriuretic peptide receptor (XNPR-C). Analysis confirms the presence of a short intracellular C-terminus, as well as a high similarity to fish and mammalian NPR-C. Injection of XNPR-C mRNA into Xenopus oocytes resulted in expression of high affinity [(125)I]ANP binding sites that were competitively and completely displaced by natriuretic analogs and the unrelated neuropeptide vasoactive intestinal peptide (VIP). Measurement of cAMP levels in mRNA-injected oocytes revealed that XNPR-C is negatively coupled to adenylyl cyclase in a pertussis toxin-sensitive manner. When XNPR-C was co-expressed with PAC(1) receptors for pituitary adenylyl cyclase-activating polypeptide (PACAP), VIP and natriuretic peptides counteracted the cAMP induction by PACAP. These results suggest that VIP and natriuretic peptides can potentially modulate the action of PACAP in cells where these receptors are co-expressed.     

Stem cell factor influences neuro-immune interactions: the response of mast cells to pituitary adenylate cyclase activating polypeptide is altered by stem cell factor.

Mast cells degranulation can be elicited by a number of biologically important neuropeptides, but the mechanisms involved in mast cell-neuropeptide interactions have not been fully elucidated. Stem cell factor (SCF), also known as c-kit or kit ligand, induces multiple effects on mast cells, including proliferation, differentiation, maturation, and prevents apoptosis. We investigated the ability of SCF to affect mast cell responsiveness to the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PACAP 1-27, PACAP1-38, or VIP failed to induced preformed mediator release from mouse bone-marrow-cultured mast cells (BMCMC) derived in concanavalin A-stimulated spleen conditioned medium (CM). By contrast, BMCMC grown in SCF-containing medium or freshly isolated peritoneal mast cells exhibited significant 3H-hydroxytrypamine (5-HT) release in response to PACAP peptides or VIP. Deoxyglucose and the mitochondrial inhibitor antimycin significantly inhibited PACAP-induced 5-HT release indicating that the central event induced by PACAP peptides was exocytosis. The G(alpha)i inhibitor, pertussis toxin, significantly diminished PACAP-induced 5-HT release from BMCMCs in SCF suggesting the involvement of heterotrimeric G-proteins. Western blot analysis using antibodies directed against the human VIP type I/PACAP type II receptor demonstrated a 70-72 kD immunoreactive protein expressed in greater amounts in BMCMC grown in SCF compared with BMCMC in CM. We conclude that SCF induces a mast cell population that is responsive to PACAPs and VIP involving a heterotrimeric G-protein-dependent mechanism.     

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP(1-38) in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide(1-38) (PACAP(1-38)), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide-SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 degrees C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP(1-38)-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP(1-38) evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 degrees C. When the incubation temperature was increased to 37 degrees C, SSM-associated PACAP(1-38)-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in alpha-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP(1-38), greater kinetic energy or longer incubation period is required to optimize peptide-SSM interactions and amplify peptide bioactivity in vivo.     

Vascular effects of pituitary adenylate cyclase activating peptide: a comparison with vasoactive intestinal peptide

The effects of pituitary adenylate cyclase activating peptide (PACAP) on the blood pressure of the anesthetized rat and on the isolated rat tail artery were investigated and compared to those of vasoactive intestinal peptide (VIP). PACAP-38, PACAP-27 and the C-terminal fragment 16–38 caused a dose-dependent decrease in the systemic blood pressure. PACAP-27 and PACAP-38 were equipotent with VIP. The C-terminal fragment 16–38 was much less potent than VIP. The duration of action was longer for equimolar doses of PACAP-38 and PACAP-27 than for VIP and much longer than for PACAP 16–38. PACAP-27 and the phosphodiesterase inhibitor rolipram given in combination produced additive vasodepressive responses. In vitro PACAP-38, PACAP-27, VIP and PACAP 16–38 relaxed the phenylephrine-precontracted rat tail artery. PACAP-38 and PACAP-27 were equipotent with VIP. PACAP 16–38 was much less potent than the full-length peptides. The responses were resistant to atropine and propranolol. Addition of VIP 1 μM to preparations exposed to 1 μM PACAP-38 or -27 did not produce a further relaxation. VIP-like peptides, PACAP in particular, are known to activate adenylate cyclase and to elevate the plasma cyclic AMP (cAMP) concentration. cAMP was found to be a potent vasodepressor in the anaesthetized rat and a potent vasodilator of precontracted blood vessels. On the basis of these results it cannot be excluded that the vascular effects of PACAP are secondary to the effect of elevated levels of extracellular cAMP.     

Early changes in pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide and related receptors expression in retina of streptozotocin-induced diabetic rats

The retinal expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) and their receptors was investigated in early streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection (60mg/kg i.p.). PACAP, VIP and their receptors in nondiabetic control and diabetic retinas were assayed by quantitative real-time PCR and Western blot 1 and 3 weeks after STZ injection. Effects of intravitreal treatment with PACAP38 on the expression of the two apoptotic-related genes Bcl-2 and p53 were also evaluated. PACAP and VIP, as well as VPAC1 and VPAC2 receptors, but not PAC1 mRNA levels, were transiently induced in retinas 1week following STZ. These findings were confirmed by immunoblot analyses. Three weeks after the induction of diabetes, significant decreases in the expression of peptides and their receptors were observed, Bcl-2 expression decreased and p53 expression increased. Intravitreal injection of PACAP38 restored STZ-induced changes in retinal Bcl-2 and p53 expression to nondiabetic levels. The initial upregulation of PACAP, VIP and related receptors and the subsequent downregulation in retina of diabetic rats along with the protective effects of PACAP38 treatment, suggest a role for both peptides in the pathogenesis of diabetic retinopathy.     

Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC(1)) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC(1) receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC(1) receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca(2+), the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: players in innate and adaptive immunity.

Recent reports identified and described neural pathways, both hard-wiring and soluble mediators, that control and adjust the peripheral immune response. Immune organs are innervated by fibers rich in neurotransmitters and neuropeptides released in inflammatory conditions. Here we focus on the immunomodulatory role of two peptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP). VIP/PACAP are present and released from both innervation and immune cells, particularly Th2 cells, and immune cells express receptors for VIP/PACAP. VIP/PACAP have a general anti-inflammatory effect, both in innate and adaptive immunity. In innate immunity, VIP/PACAP inhibit the production of pro-inflammatory cytokines and chemokines from macrophages, microglia and dendritic cells. In addition, VIP/PACAP reduce the expression of costimulatory molecules (particularly CD80 and CD86) on the antigen-presenting cells, and therefore reduce stimulation of antigen-specific CD4+ T cells. In terms of adaptive immunity, VIP/PACAP promote Th2-type responses, and reduce the pro-inflammatory Th1-type responses. Several of the molecular mechanisms involved in the inhibition of cytokine and chemokine expression, and in the preferential development and/or survival of Th2 effects, are discussed.     

VIP and PACAP display different vasodilatory effects in rabbit coronary and cerebral arteries

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are closely related peptides with wide distribution in the nervous system. The aim of the present study was to investigate functional characteristics and the influence of sex steroids on the vasodilatory effects of these two peptides in cerebral and coronary vessels from female New Zealand White (NZW) rabbits.

The localization and concentration of VIP and PACAP in cardiovascular tissue was evaluated using immunohistochemistry and radioimmunoassays. The vasodilatory effects of VIP and PACAP were investigated using myographs, allowing isometric tension recordings. In order to evaluate the influence of steroid hormones, the rabbits were ovariectomized and randomized to treatment for 4 weeks with 17β-estradiol (E₂), Norethindrone Acetate (NETA), E₂+NETA or placebo. Ring segments of the posterior cerebral artery, the right proximal coronary artery and the distal left coronary artery were examined.

The highest concentrations of VIP/PACAP were observed in cerebral and coronary arteries: 5.0/5.7 and 2.8/3.5 pmol/g, respectively. The peptides were localized in nerve fibres innervating the arteries. Both peptides produced dose-dependent vasodilatory responses in all vessels investigated. While the effects of PACAP were identical in cerebral and coronary arterial segments, the effects of VIP displayed significant differences (E_max, pI₂, Hill-slope). Treatment with sex steroids induced no changes in the vascular effects of the two peptides.

These results indicate different mechanisms of action for the vasodilating effects of the two closely related peptides VIP and PACAP in different areas of the coronary and cerebrovascular tree. Treatment with female sex steroids does not seem to change these mechanisms.     

PACAP and VIP increase the expression of myelin-related proteins in rat schwannoma cells: Involvement of PAC1/VPAC2 receptor-mediated activation of PI3K/Akt signaling pathways

PACAP and its cognate peptide VIP participate in various biological functions, including myelin maturation and synthesis. However, defining whether these peptides affect peripheral expression of myelin proteins still remains unanswered. To address this issue, we assessed whether PACAP or VIP contribute to regulate the expression of three myelin proteins (MAG, MBP and MPZ, respectively) using the rat schwannoma cell line (RT4-P6D2T), a well-established model to study myelin gene expression. In addition, we endeavored to partly unravel the underlying molecular mechanisms involved. Expression of myelin-specific proteins was assessed in cells grown either in normal serum (10% FBS) or serum starved and treated with or without 100 nM PACAP or VIP. Furthermore, through pharmacological approach using the PACAP/VIP receptor antagonist (PACAP6-38) or specific pathway (MAPK or PI3K) inhibitors we defined the relative contribution of receptors and/or signaling pathways on the expression of myelin proteins. Our data show that serum starvation (24 h) significantly increased both MAG, MBP and MPZ expression. Concurrently, we observed increased expression of endogenous PACAP and related receptors. Treatment with PACAP or VIP further exacerbated starvation-induced expression of myelin markers, suggesting that serum withdrawal might sensitize cells to peptide activity. Stimulation with either peptides increased phosphorylation of Akt at Ser473 residue but had no effect on phosphorylated Erk-1/2. PACAP6-38 (10 μM) impeded starvation- or peptide-induced expression of myelin markers. Similar effects were obtained after pretreatment with the PI3K inhibitor (wortmannin, 10 μM) but not the MAPKK inhibitor (PD98059, 50 μM). Together, the present finding corroborate the hypothesis that PACAP and VIP might contribute to the myelinating process preferentially via the canonical PI3K/Akt signaling pathway, providing the basis for future studies on the role of these peptides in demyelinating diseases.     

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.     

High levels of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor mRNA expression in primary and tumor lymphoid cells

Neuropeptides exert a variety of putative immunomodulatory actions. Despite the molecular cloning of multiple forms of receptors for several neuropeptides with putative immunomodulatory effects, including vasoactive intestinal peptide (VIP), the related peptide pituitary adenylate cyclase-activating peptide (PACAP), the opiate peptides, tachykinins, somatostatin and corticotropin-releasing factor, it has not been reported that any of the receptor genes are expressed at significant levels in cells of the immune system. The low level of expression of these receptors and lack of knowledge concerning receptor subtype has impeded progress in understanding how neuropeptides regulate immune function. For example, it is not understood why VIP produces immunomodulatory effects at concentrations far below its receptor-binding affinity. Receptors for VIP and PACAP have recently been cloned. We show here by Northern blot analysis that the VIP/PACAP₁ receptor mRNA is present in total RNA prepared from mouse spleen B- and T-lymphocytes. The VIP/PACAP₁ receptor mRNA was also present in human peripheral blood lymphocytes, and in a B-lymphocyte and a myelocytic cell line. The mRNA for a second form of the receptor, the VIP/PACAP₂ receptor, was not expressed at detectable levels in normal cells, but was detected in several human T-cell lines and a murine mast cell line. The results indicate that VIP/PACAP₁ and perhaps VIP/PACAP₂ receptors mediate the diverse effects of VIP and PACAP on immune cells.     

Neuroprotective roles of pituitary adenylate cyclase-activating polypeptide in neurodegenerative diseases

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic bioactive peptide that was first isolated from an ovine hypothalamus in 1989. PACAP belongs to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily. PACAP is widely distributed in the central and peripheral nervous systems and acts as a neurotransmitter, neuromodulator, and neurotrophic factor via three major receptors (PAC1, VPAC1, and VPAC2). Recent studies have shown a neuroprotective role of PACAP using in vitro and in vivo models. In this review, we briefly summarize the current findings on the neurotrophic and neuroprotective effects of PACAP in different brain injury models, such as cerebral ischemia, Parkinson’s disease (PD), and Alzheimer’s disease (AD). This review will provide information for the future development of therapeutic strategies in treatment of these neurodegenerative diseases. [BMB Reports 2014; 47(7): 369-375]     

Local Effect of PACAP and VIP on Testicular Function in Immature and Adult Rats

Csaba, Zs., V. Csernus, and I. Gerendai. Local effect of PACAP and VIP on testicular function in immature and adult rats. Peptides 18(10) 1561–1567, 1997.—PACAP, VIP, anti-PACAP and anti-VIP antisera were injected intratesticularly. In 9-day-old hemicastrated rats PACAP or VIP decreased basal testosterone secretion. In 22-day-old hemicastrates VIP but not PACAP reduced compensatory testicular hypertrophy, however, neither PACAP nor VIP altered steroidogenesis. Anti-VIP antiserum to this age group increased testosterone production and enhanced compensatory testicular hypertrophy. In adult hemicastrates neither the peptides nor the antisera influenced steroidogenesis. Neither in immatures nor in adults treatment of both testes with PACAP or VIP had any effect. Data indicate that both PACAP and VIP might exert a local action on testicular steroidogenesis, on compensatory testicular hypertrophy, and these effects are age-dependent.     

PACAP and a novel stable analog protect rat brain from ischemia: Insight into the mechanisms of action

Pituitary adenylate cyclase-activating polypeptide (PACAP) shows potent protective effects in numerous models of neurological insults. However, the use of PACAP as a clinically efficient drug is limited by its poor metabolic stability. By combining identification of enzymatic cleavage sites with targeted chemical modifications, a metabolically stable and potent PACAP38 analog was recently developed. The neuroprotective activity of this novel compound was for the first time evaluated and compared to the native peptide using a rat model of middle cerebral artery occlusion (MCAO). Our results show that as low as picomolar doses of PACAP38 and its analog strongly reduce infarct volume and improve neurological impairment induced by stroke. In particular, these peptides inhibit the expression of Bcl-2-associated death promoter, caspase 3, macrophage inflammatory protein-1α, inducible nitric oxide synthase 2, tumor necrosis factor-α mRNAs, and increase extracellular signal-regulated kinase 2, B-cell CLL/lymphoma 2 and interleukin 6 mRNA levels. These results indicate that the neuroprotective effect of PACAP after MCAO is not only due to its ability to inhibit apoptosis but also to modulate the inflammatory response. The present study highlights the potential therapeutic efficacy of very low concentrations of PACAP or its metabolically stable derivative for the treatment of stroke.     

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).