Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of 'tyrosine toggle switch' required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist - cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

Synthesis and In Vitro Characterization of Glycopeptide Drug Candidates Related to PACAP1-23.

The search for efficacious treatment of neurodegenerative and progressive neuroinflammatory diseases continues, as current therapies are unable to halt or reverse disease progression. PACAP represents one potential therapeutic that provides neuroprotection effects on neurons, and also modulates inflammatory responses and circulation within the brain. However, PACAP is a relatively long peptide hormone that is not trivial to synthesize. Based on previous observations that the shortened isoform PACAP1-23 is capable of inducing neuroprotection in vitro, we were inspired to synthesize shortened glycopeptide analogues of PACAP1-23. Herein, we report the synthesis and in vitro characterization of glycosylated PACAP1-23 analogues that interact strongly with the PAC1 and VPAC1 receptors, while showing reduced activity at the VPAC2 receptor.

Mechanisms for establishment of GABA signaling in adrenal medullary chromaffin cells.

γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABAA receptor Cl- channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABAA receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABAA receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABAA receptor Cl- channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABAA receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABAA receptors and GAD67 involved in GABA signaling in AMC cells, respectively.

Inhibition of PACAP/PAC1/VPAC2 signaling impairs the consolidation of social recognition memory and nitric oxide prevents this deficit.

Social recognition memory (SRM) forms the basis of social relationships of animals. It is essential for social interaction and adaptive behavior, reproduction and species survival. Evidence demonstrates that social deficits of psychiatric disorders such as autism and schizophrenia are caused by alterations in SRM processing by the hippocampus and amygdala. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptors PAC1, VPAC1 and VPAC2 are highly expressed in these regions. PACAP is a pleiotropic neuropeptide that modulates synaptic function and plasticity and is thought to be involved in social behavior. PACAP signaling also stimulates the nitric oxide (NO) production and targets outcomes to synapses. In the present work, we investigate the effect of the infusion of PACAP-38 (endogenous neuropeptide and potent stimulator of adenylyl cyclase), PACAP 6-38 (PAC1/VPAC2 receptors antagonist) and S-Nitroso-N-acetyl-DL-penicillamine (SNAP, NO donor) in the CA1 region of the hippocampus and in the basolateral amygdala (BLA) on the consolidation of SRM. For this, male Wistar rats with cannulae implanted in CA1 or in BLA were subjected to a social discrimination paradigm, which is based on the natural ability of rodents to investigate unfamiliar conspecifics more than familiar one. In the sample phase (acquisition), animals were exposed to a juvenile conspecific for 1 h. Immediately, 60 or 150 min after, animals received one of different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. Animals that received infusions of PACAP 6-38 (40 pg/side) into CA1 immediately after the sample phase or into BLA immediately or 60 min after the sample phase were unable to recognize the familiar juvenile during the retention test. This impairment was abolished by the coinfusion of PACAP 6-38 plus SNAP (5 µg/side). These results show that the blockade of PACAP/PAC1/VPAC2 signaling in the CA1 and BLA during a restricted post-acquisition time window impairs the consolidation of SRM and that the SNAP is able to abolish this deficit. Findings like this could potentially be used in the future to influence studies of psychiatric disorders involving social behavior.

Pituitary Adenylate Cyclase-Activating Polypeptide Excites Proopiomelanocortin Neurons: Implications for the Regulation of Energy Homeostasis.

OBJECTIVE: We examined whether pituitary adenylate cyclase-activating polypeptide (PACAP) excites proopiomelanocortin (POMC) neurons via PAC1 receptor mediation and transient receptor potential cation (TRPC) channel activation. METHODS: Electrophysiological recordings were done in slices from both intact male and ovariectomized (OVX) female PACAP-Cre mice and eGFP-POMC mice. RESULTS: In recordings from POMC neurons in eGFP-POMC mice, PACAP induced a robust inward current and increase in conductance in voltage clamp, and a depolarization and increase in firing in current clamp. These postsynaptic actions were abolished by inhibitors of the PAC1 receptor, TRPC channels, phospholipase C, phosphatidylinositol-3-kinase, and protein kinase C. Estradiol augmented the PACAP-induced inward current, depolarization, and increased firing, which was abrogated by estrogen receptor (ER) antagonists. In optogenetic recordings from POMC neurons in PACAP-Cre mice, high-frequency photostimulation induced inward currents, depolarizations, and increased firing that were significantly enhanced by Gq-coupled membrane ER signaling in an ER antagonist-sensitive manner. Importantly, the PACAP-induced excitation of POMC neurons was notably reduced in obese, high-fat (HFD)-fed males. In vivo experiments revealed that intra-arcuate nucleus (ARC) PACAP as well as chemogenetic and optogenetic stimulation of ventromedial nucleus (VMN) PACAP neurons produced a significant decrease in energy intake accompanied by an increase in energy expenditure, effects blunted by HFD in males and partially potentiated by estradiol in OVX females. CONCLUSIONS: These findings reveal that the PACAP-induced activation of PAC1 receptor and TRPC5 channels at VMN PACAP/ARC POMC synapses is potentiated by estradiol and attenuated under conditions of diet-induced obesity/insulin resistance. As such, they advance our understanding of how PACAP regulates the homeostatic energy balance circuitry under normal and pathophysiological circumstances.

The role of pituitary adenylyl cyclase-activating polypeptide in the motivational effects of addictive drugs.

Pituitary adenylyl cyclase activating polypeptide (PACAP) was originally isolated from the hypothalamus and found to stimulate adenylyl cyclase in the pituitary. Later studies showed that this peptide and its receptors (PAC1, VPAC1, and VPAC2) are widely expressed in the central nervous system (CNS). Consistent with its distribution in the CNS, the PACAP/PAC1 receptor system is involved in several physiological responses, such as mediation of the stress response, modulation of nociception, regulation of prolactin release, food intake, etc. This system is also implicated in different pathological states, e.g., affective component of nociceptive processing, anxiety, depression, schizophrenia, and post-traumatic stress disorders. A review of the literature on PubMed revealed that PACAP and its receptors also play a significant role in the actions of addictive drugs. The goal of this review is to discuss the literature regarding the involvements of PACAP and its receptors in the motivational effects of addictive drugs. We particularly focus on the role of this peptide in the motivational effects of morphine, alcohol, nicotine, amphetamine, methamphetamine, and cocaine. This article is part of the special issue on Neuropeptides.

Effects of PACAP-38 and an analog, acetyl-[Ala15, Ala20] PACAP-38-propylamide, on memory consolidation in the detection of spatial novelty task in rats.

PACAP-38 (P38) is a pleiotropic peptide that exerts multiple peripheral and central actions, including neurotrophic, neuroprotective and anti-inflammatory actions. Previous studies have suggested an improvement of memory in rats that have received a single systemic injection of P38. In a therapeutic perspective, we used an analog, acetyl-[Ala15, Ala20]PACAP-38-propylamide (ALG), to improve both stability and affinity for PAC1 receptors vs. endogen PACAP. We investigated the effect of P38 and ALG on memory consolidation using a spatial novelty detection (SND) task in which rats had to memorize a configuration of objects to identify that, during a test session, a familiar object has been moved to a new location. Rats received an intravenous injection of P38 or ALG after the last training session. In Experiment 1, P38 (30 µg/kg) improved spatial memory consolidation allowing detection of novelty vs. saline injection. In Experiment 2, we confirmed this effect and showed that P38 restored the performance similar to what was found using non-injected rats. This suggests that, contrary to ALG, P38 exerted a promesiant rather than an anxiety-related effect whereas ALG did not show similar action. We also examined whether P38 effect involved an interaction with NR2B-containing NMDA receptors (NMDARs) by administrating ifenprodil (IFE; a selective NR2B-containing NMDAR antagonist) alone or in combination with P38 or ALG. The results suggested that P38 action on memory involved NR2B-containing NMDARs. Lastly, brain-derived neutrophic factor (BDNF) modulation appeared to be not related to the behavioral performance in the SND task. Overall, the results indicate that P38 exerted a beneficial effect on memory consolidation in a non-associative task, whereas ALG did not have this action.

PACAP-(6-38) or kynurenate microinjections in the RVLM prevent the development of sympathetic long-term facilitation after acute intermittent hypoxia.

Intermittent hypoxia causes a persistent increase in sympathetic activity that progresses to hypertension in chronic conditions such as obstructive sleep apnea. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neurotransmitter that causes long-lasting sympathetic excitation. We aimed to determine if intermittent activation of the rostral ventrolateral medulla (RVLM) causes PACAP-mediated elevation of sympathetic nerve activity, termed sympathetic long-term facilitation (sLTF). The role of PACAP in mediating sLTF in response to intermittent activation of the RVLM was investigated in urethane-anaesthetized and artificially ventilated rats ( n = 65, Sprague-Dawley). Bilateral RVLM microinjections of the PACAP type 1 receptor/vasoactive intestinal polypeptide receptor type 2 receptor antagonist PACAP-(6-38) [ n = 6, change (Δ): -16.4 ± 6.5%) or an ionotropic glutamate antagonist, kynurenate ( n = 6, Δ:-7.2 ± 2.3%), blocked the development of acute intermittent hypoxia-induced sLTF ( n = 6, Δ: 49.2 ± 14.2%). Intermittent RVLM microinjections of glutamate caused sLTF ( n = 5, Δ: 56.9 ± 14.7%) that was abolished by PACAP-(6-38) pretreatment ( n = 5, Δ:-1.2 ± 4.7%). Conversely, intermittent microinjections of PACAP in the RVLM did not elicit sLTF. Intermittent bilateral disinhibition of the RVLM by microinjection of γ-aminobutyric acid in the caudal ventrolateral medulla did not elicit sLTF. Direct activation of RVLM neurons is crucial for the development of sLTF. PACAP and glutamate act synergistically in the RVLM, with both being necessary for the sLTF response. We found that activation of glutamate but not PACAP receptors is necessary and sufficient to generate sLTF, even in the absence of intermittent hypoxia. Our results demonstrate that PACAP within the RVLM may contribute to the development of obstructive sleep apnea -induced hypertension. NEW & NOTEWORTHY Pharmacological blockade of either pituitary adenylate cyclase-activating polypeptide (PACAP) or ionotropic glutamate receptors in the rostral ventrolateral medulla prevents development of sympathetic long-term facilitation. PACAP receptor inhibition prevents the occurrence of hypoxia-induced peripheral chemoreflex sensitization. Thus, PACAP receptors may be a potential therapeutic target serving to reduce heightened sympathetic tone and hypersensitized cardiovascular reflexes.

PACAP receptor pharmacology and agonist bias: analysis in primary neurons and glia from the trigeminal ganglia and transfected cells.

BACKGROUND AND PURPOSE: A major challenge in the development of new medicines targeting GPCRs is the ability to quantify drug action in physiologically relevant models. Primary cell models that closely resemble the clinically relevant in vivo site of drug action are important translational tools in drug development. However, pharmacological studies in these models are generally very limited due to the methodology used. EXPERIMENTAL APPROACH: We used a neuropeptide system to demonstrate the applicability of using highly sensitive signalling assays in primary cells. We quantified the action of pituitary adenylate cyclase-activating peptide (PACAP)-38, PACAP-27 and vasoactive intestinal polypeptide in primary cultures of neurons and glia derived from rat trigeminal ganglia (TG), comparing our observations to transfected cells. KEY RESULTS: PACAP-responsive receptors in rat trigeminal neurons, glia and transfected PAC1n receptors were pharmacologically distinct. PACAP-38, but not PACAP-27, activated ERK in glia, while both forms stimulated cellular cAMP production. PACAP(6-38) also displayed cell-type-dependent, agonist-specific, antagonism. CONCLUSIONS AND IMPLICATIONS: The complexity of PACAP pharmacology in the TG may help to direct, more effectively, the development of disease treatments targeting the PACAP receptor. We suggest that these methodologies are broadly applicable to other primary cell types of human or animal origin, and that our approach may allow more thorough characterization of ligand properties in physiologically relevant cell types.

Interaction of gonadal steroids and gonadotropin-releasing hormone on pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptor expression in cultured rat anterior pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine-paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH.

VIP/PACAP receptors in cerebral arteries of rat: characterization, localization and relation to intracellular calcium.

BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)-containing nerves surround cerebral blood vessels. The peptides have potent vasodilator properties via smooth muscle cell receptors and activation of adenylate cyclase. The purpose of this study was to describe the effects of two putative VIP/PACAP receptor antagonists and the distribution of the receptor protein in rat brain vessels. METHODS: The vascular effects of VIP, PACAP-27 and PACAP-38 were investigated in segments of rat middle cerebral artery (MCA) by pressurized arteriography, and in a wire myograph. The antagonistic responses to PACAP6-38 and PG99-465 were evaluated. In addition, the receptor subtypes for VIP and PACAP (VPAC1, VPAC2 and PAC1) were visualized in the rat middle cerebral artery by immunohistochemistry and Western blotting. RESULTS: In the perfusion model, abluminal but not luminal VIP, PACAP-27 and PACAP-38 caused concentration-dependent relaxations of the MCA (27.1±0.2%, 25.2±0.4% and 0.3±0.1%, respectively). In the wire myograph, there was no significant difference in potency of the peptides in the MCA. In both systems, PACAP6-38 and PG99-465 inhibited the VIP induced relaxation. Western blot showed the presence of the receptor proteins in cerebral vasculature and immunohistochemistry showed that all three receptors are present and located in the cytoplasm of smooth muscle cells. CONCLUSION: In both systems, the two blockers antagonized the relaxant VIP effect; the potency order of agonists and the immunohistochemistry suggest the presence of the dilatory VPAC1 and VPAC2 receptors on the smooth muscle cells.

Multimodal neuroprotection induced by PACAP38 in oxygen-glucose deprivation and middle cerebral artery occlusion stroke models.

Pituitary adenylate cyclase activating peptide (PACAP), a potent neuropeptide which crosses the blood-brain barrier, is known to provide neuroprotection in rat stroke models of middle cerebral artery occlusion (MCAO) by mechanism(s) which deserve clarification. We confirmed that following i.v. injection of 30 ng/kg of PACAP38 in rats exposed to 2 h of MCAO focal cerebral ischemia and 48 h reoxygenation, 50 % neuroprotection was measured by reduced caspase-3 activity and volume of cerebral infarction. Similar neuroprotective effects were measured upon PACAP38 treatment of oxygen-glucose deprivation and reoxygenation of brain cortical neurons. The neuroprotection was temporally associated with increased expression of brain-derived neurotrophic factor, phosphorylation of its receptor-tropomyosin-related kinase receptor type B (trkB), activation of phosphoinositide 3-kinase and Akt, and reduction of extracellular signal-regulated kinases 1/2 phosphorylation. PACAP38 increased expression of neuronal markers beta-tubulin III, microtubule-associated protein-2, and growth-associated protein-43. PACAP38 induced stimulation of Rac and suppression of Rho GTPase activities. PACAP38 downregulated the nerve growth factor receptor (p75(NTR)) and associated Nogo-(Neurite outgrowth-A) receptor. Collectively, these in vitro and in vivo results propose that PACAP exhibits neuroprotective effects in cerebral ischemia by three mechanisms: a direct one, mediated by PACAP receptors, and two indirect, induced by neurotrophin release, activation of the trkB receptors and attenuation of neuronal growth inhibitory signaling molecules p75(NTR) and Nogo receptor.

PYK-2 is tyrosine phosphorylated after activation of pituitary adenylate cyclase activating polypeptide receptors in lung cancer cells.

The signal transduction mechanisms of pituitary adenylate cyclase activating polypeptide (PACAP) were investigated in lung cancer cells. Previously, PACAP-27 addition to NCI-H838 cells increased phosphatidylinositol turnover and intracellular cAMP leading to proliferation of lung cancer cells. Also, PACAP receptors (PAC1) regulated the tyrosine phosphorylation of ERK, focal adhesion kinase, and paxillin. In this communication, the effects of PACAP on cytosolic Ca(2+) and PYK-2 tyrosine phosphorylation were investigated. PACAP-27 increased cytosolic Ca(2+) within seconds after addition to FURA-2 AM loaded NCI-H838 cells. The increase in cytosolic Ca(2+) caused by PACAP was inhibited by PACAP(6-38) (PAC1 antagonist), U73122 (phospholipase C inhibitor), or BAPTA (calcium chelator), but not H89 (PKA inhibitor). PACAP-38, but not vasoactive intestinal peptide (VIP), addition to NCI-H838 or H1299 cells significantly increased the tyrosine phosphorylation of PYK-2 after 2 min. The increase in PYK-2 tyrosine phosphorylation caused by PACAP was inhibited by PACAP(6-38), U73122, or BAPTA, but not H89. The results suggest that PAC1 regulates PYK-2 tyrosine phosphorylation in a calcium-dependent manner.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1-CXCR1/2-pathway.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

A novel cyclopeptide from the cyclization of PACAP(1-5) with potent activity towards PAC1 attenuates STZ-induced diabetes.

The N-terminal deletion of pituitary adenylate cyclase-activating polypeptide (PACAP)(1-5) generates its own antagonist. The cyclopeptide C*HSDGIC*, which results from the cyclization of PACAP(1-5) with disulfide, was designed and synthesized. CHO cells expressing a PAC1 N/R splice variant (PAC1-CHO) were used to detect the potent activation of PAC1 by C*HSDGIC*. In vitro cell assays showed that C*HSDGIC* stimulated cAMP production and increased the viability of PAC1-CHO cells at micromolar concentrations, about 1000 fold that of PACAP. PACAP(6-38) blocked the effects of PACAP on the proliferation of PAC1-CHO cells but did not interfere with the effects of C*HSDGIC*, suggesting that the activation of PAC1 by C*HSDGIC* was independent of the binding of PAC1 to the C-terminus of PACAP. In experiments in vivo, 10mumol/kg C*HSDGIC* decreased the plasma glucose level, increased the plasma insulin level and improved glucose tolerance significantly (P<0.01) when co-injected with STZ for 5 days. The results of these in vitro and in vivo studies of the biological characteristics of C*HSDGIC* reveal that it is a potent activator of PAC1.

Identification and characterization of a small molecule antagonist of human VPAC(2) receptor.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) and their class II G protein-coupled receptors VPAC(1), VPAC(2), and PAC(1) play important roles in human physiology. No small molecule modulator has ever been reported for the VIP/PACAP receptors, and there is a lack of specific VPAC(2) antagonists. Via high-throughput screening of 1.67 million compounds, we discovered a single small molecule antagonist of human VPAC(2), compound 1. Compound 1 inhibits VPAC(2)-mediated cAMP accumulation with an IC(50) of 3.8 microM and the ligand-activated beta-arrestin2 binding with an IC(50) of 2.3 microM. Compound 1 acts noncompetitively in Schild analysis. It is a specific VPAC(2) antagonist with no detectable agonist or antagonist activities on VPAC(1) or PAC(1). Compound 2, a close structural analog of compound 1, was also found to be weakly active. To our surprise, compound 1 is completely inactive on the closely related mouse VPAC(2). Chimera experiments indicate that compounds 1 and 2 bind to the seven transmembrane (7TM) region of the receptor as opposed to the N-terminal extracellular domain, where the natural ligand binds. Compound 1, being the first small molecular antagonist that is specific for VPAC(2), and the only VPAC(2) antagonist molecule known to date that allosterically interacts with the 7TM region, will be a valuable tool in further study of VPAC(2) and related receptors. This study also highlights the opportunities and challenges facing small molecule drug discovery for class II peptide G protein-coupled receptors.

PACAP attenuates 5-HT, histamine, and ATP-evoked Ca2+ transients in astrocytes.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective properties and plays an important role in neuroinflammation. PACAP38 interacts with its receptors, PAC1, and VPAC, on astrocytes at 10(-8) M to induce biphasic Ca2+ transients, which were reduced to a single transient by the PAC1-blocking PACAP antagonist PACAP6-38. At 10(-12) M even the single transient, corresponding to PAC1 was blocked. PACAP-induced Ca2+ transients were more pronounced in astrocytes cocultured with brain endothelial cells than in monocultured astrocytes, indicating that astrocytes that receive signals from microvessels develop more sensitive signal transduction systems for Ca. In this sensitive system, PACAP38 attenuated 5-HT, histamine, and ATP-evoked Ca2+ transients, showing the anti-inflammatory properties of PACAP.

Role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the action of ginsenoside Rh2 against beta-amyloid-induced inhibition of rat brain astrocytes.

Ginsenoside, the active principles in Panax ginseng root, has been demonstrated to show neurotrophic and neuroprotective actions for prevention of neuron degeneration. Deposition of beta-amyloid peptide (Abeta) causes neurotoxicity through the formation of plaques in brains with Alzheimer's disease. Pituitary adenylate cyclase-activating polypeptide (PACAP) is introduced as a neurotrophic factor to promote cell survival. However, effect of Rh2, one of ginsenosides, on PACAP expression induced by Abeta remains unclear. In the present study, we found that Rh2 stimulates PACAP gene expression and cell proliferation in type I rat brain astrocytes (RBA1) cells and both effects were not modified by the estrogen antagonists (MPP or ICI 182780). Also, Rh2 ameliorates the RBA1 growth inhibition of Abeta. Moreover, blockade of PACAP receptor PAC1 using PACAP (6-38) inhibits all the actions of Rh2. These results suggest that Rh2 can induce an increase of PACAP to activate PAC1, but not estrogen receptor, and thereby leads to attenuate Abeta-induced toxicity. Thus, ginseng seems useful in the prevention of dementia.

Novel stable PACAP analogs with potent activity towards the PAC1 receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.