Vasoactive Intestinal Peptide Receptor Research Articles

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.

β-Arrestin-independent endosomal cAMP signaling by a polypeptide hormone GPCR

Many G protein-coupled receptors (GPCRs) initiate a second phase of stimulatory heterotrimeric G protein (Gs)-coupled cAMP signaling after endocytosis. The prevailing current view is that the endosomal signal is inherently β-arrestin-dependent because β-arrestin is necessary for receptor internalization and, for some GPCRs, to prolong the endosomal signal. Here we revise this view by showing that the vasoactive intestinal peptide receptor 1 (VIPR1), a secretin-family polypeptide hormone receptor, does not require β-arrestin to internalize or to generate an endosomal signal. β-Arrestin instead resolves the plasma membrane and endosomal signaling phases into sequential cAMP peaks by desensitizing the plasma membrane phase without affecting the endosomal phase. This appears to occur through the formation of functionally distinct VIPR1–β-arrestin complexes at each location that differ in their phosphorylation dependence. We conclude that endosomal GPCR signaling can occur in the absence of β-arrestin and that β-arrestin sculpts the spatiotemporal profile of cellular GPCR–G protein signaling through location-specific remodeling of GPCR–β-arrestin complexes.

Neural targets of the enteric dopaminergic system in regulating motility of rat proximal colon.

In isolated segments of the rat proximal colon, the dopamine reuptake inhibitor GBR 12909 (GBR) causes a dilatation, while the D1-like receptor antagonist SCH 23390 (SCH) induces a tonic constriction, suggesting that neurally released dopamine tonically stimulates enteric inhibitory efferent neurons. Here, the targets of the enteric dopaminergic neurons were investigated. Cannulated segments of rat proximal colon were bathed in physiological salt solution and luminally perfused with 0.9% saline, while all drugs were applied to the bath. Spatio-temporal maps of colonic motility were constructed from video recordings of peristaltic contractions, and the maximum diameter was measured as an index of colonic contractility. GBR (1μM)-induced dilatations of colonic segments were prevented by SCH (5μM), L-nitro arginine (L-NA; 100μM), a nitric oxide synthase inhibitor, or tetrodotoxin (0.6μM). In contrast, constrictions induced by a higher concentration of SCH (20μM) were unaffected by either L-NA or tetrodotoxin. The vasoactive intestinal peptide (VIP) receptor antagonist VIP10-28 (3μM) or P2Y1 receptor antagonist MRS 2500 (1μM) had no effect on either the GBR-induced dilatation or the SCH-induced constriction. In colonic segments that had been pretreated with 6-hydroxydopamine (100μM, 3h) to deplete enteric dopamine, GBR failed to increase the colonic diameter, while SCH was still capable of constricting colonic segments. Enteric dopaminergic neurons appear to project to nitrergic neurons to dilate the proximal colon by activating neuronal D1-like receptors. In addition, constitutively activated D1-like receptors expressed in cells yet to be determined may provide a tonic inhibition on colonic constrictions.

Association of vasoactive intestinal peptide receptor 1 gene polymorphism with layer economic traits in Rhode Island Red chicken

The research work was conducted to determine allelic polymorphism of vasoactive intestinal peptide receptor-1 (VIPR-1) gene and its association with layer traits in Rhode Island Red (RIR) chicken. Blood samples were collected from pullets (n=111) belonging to five hatches. Data on body weight at 20 weeks of age (BW20) and layer economic traits, viz. age at sexual maturity (ASM), egg weight at 28 (EW28) and 40 weeks (EW40) of age, and egg production up to 40 weeks of age (EP40)) were recorded. Genomic DNA was isolated by phenol chloroform extraction method. Samples were amplified for product sizes of 364, 434 and 486 bp of VIPR-1 gene. Amplified regions were digested by TaiI, HhaI and TaqI restriction enzymes. Digested fragments of TaiI and HhaI revealed monomorphism, whereas TaqI showed polymorphism. Allelic frequencies for polymorphic locus were 0.98 (A) and 0.02 (B) and genotypic frequencies for AA and AB were 0.96 and 0.04, respectively. The BW20 and layer traits (ASM, EW28, EW40 and EP40) were analysed by least-squares analysis of variance taking sire as random and hatch as fixed effect. Average ASM and egg production up to 40 weeks of age were 135.19±1.15 days and 124.55±1.94 eggs, respectively. Least-square analysis of variance had revealed significant effect of hatch on ASM, EW28 and EP40. However, effect of TaqI-RFLP genotypes was non-significant on layer traits. Further investigations on a larger sample basis are suggested to confirm effect of this mutation on layer traits as molecular marker in RIR chicken.

The VIPR2-selective antagonist KS-133 changes macrophage polarization and exerts potent anti-tumor effects as a single agent and in combination with an anti-PD-1 antibody.

We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide-VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors.

Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide’s stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide’s stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors’ conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets. Communicated by Ramaswamy H. Sarma

Development of the vasoactive intestinal peptide receptor 2 (VIPR2) antagonist peptide for the treatment of schizophrenia

Schizophrenia affects approximately 24 million people worldwide. Existing medications for the treatment of schizophrenia work primarily by improving positive symptoms such as agitation, hallucinations, delusions, and aggression. They possess common mechanism of action (MOA), blocking to neurotransmitter receptors such as dopamine, serotonin, and adrenaline receptors. Although multiple agents are available for the treatment of schizophrenia, the majority do not address negative symptoms or cognitive dysfunction. In other cases, patients have drug-related adverse effects. The vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2 receptor) might be an attractive drug target for the treatment of schizophrenia because both clinical and preclinical studies have demonstrated a strong link between high expression/overactivation of VIPR2 and schizophrenia. Despite these backgrounds, the proof-of-concept of VIPR2 inhibitors has not been examined clinically. A reason might be that VIPR2 belongs to class-B GPCRs, and the discovery of small-molecule drugs against class-B GPCRs is generally difficult. We have developed a bicyclic peptide KS-133, which shows VIPR2 antagonist activity and suppresses cognitive decline in a mouse model relevant to schizophrenia. KS-133 has a different MOA from current therapeutic drugs and exhibits high selectivity for VIPR2 and potent inhibitory activity against a single-target molecule. Therefore, it may contribute to both the development of a novel drug candidate for the treatment of psychiatric disorders such as schizophrenia and acceleration of basic studies on VIPR2.

Ion transport basis of norovirus diarrhea in a neonatal mouse model

Background: Human Norovirus (HuNoV) is the leading cause of infectious gastroenteritis and food-borne outbreaks, responsible for over 220K deaths annually. Given difficulties studying HuNoV, Murine Norovirus (MNV) has been used as a model for norovirus (NoV) pathophysiology. A major barrier to understand how NoV causes diarrhea is the absence of diarrhea in adult wild-type (WT) mice, which have asymptomatic MNV infections. While several KO mouse strains (e.g., STAT1) have been employed to recapitulate disease, these mice often have exaggerated, systemic, and lethal disease courses that imperfectly match human disease. Very recently, a new model of MNV that appears to recapitulate symptomatic HuNoV infection has been established. This model involves the oral gavage of three- or four-day-old (d.o.) WT BALB/c mice with high-concentrations of MNV resulting in a diarrheal phenotype. Current studies were undertaken to understand the ion transport basis of MNV diarrhea using this novel model. Methods: Three to four d.o. BALB/c pups were gavaged with either 1x10 8 plaque-forming units (PFU) of MNV-1 (CW3) or PBS alone followed by sacrifice at 48 hours-post-gavage (hpg). Small and large intestines were harvested for RNA and immunofluorescence (IF) analysis of multiple ion transporters that have been linked to diarrhea in other disease models. Results: MNV-gavaged pups had loose, orange stools, consistent with a diarrheal phenotype observed previously with this model. MNV RNA was detectable in small and large intestinal tissue for CW3-gavaged vs mock-gavaged pups (p-values <0.05) demonstrating successful infection with MNV. MNV infection had no significant effect on transcript levels of various electrolyte and nutrient transporters including DRA, NHE3, PAT-1, Na-K-ATPase, or SGLT-1. However, we found significant increases in CFTR and Vasoactive intestinal peptide receptor 1 (VPAC1) transcripts (p <0.05 and 0.01, respectively) and a significant increase in CFTR IF staining in frozen sections of the SI over mock (p<0.01). We did not find a significant increase in VIP transcript, VPAC1 protein expression or immunofluorescent staining, nor nuclear localization of pCREB in SI frozen sections. Conclusion: Using this novel neonatal WT BALB/c MNV mouse model to evaluate the effect of infection on gene transcription, and protein expression and localization of ion transporters commonly implicated in diarrhea, we observed significant increases in CFTR and VPAC1 SI gene expression in CW3-gavaged pups with negligible changes in transcription of all other transporters examined in this study. In addition, apical and total CFTR staining increased significantly without changes in VPAC1 or pCREB nuclear localization in frozen SI sections of CW3-gavaged pups compared to mock, suggesting that MNV causes a CFTR-mediated secretory diarrhea in the absence of changes in VPAC1 protein expression, localization, or activation. These studies were supported by the Department of Veterans Affairs, Veterans Heath Administration, Office of Research and Development, Biomedical Laboratory Research and Development: Merit Review Award: BX002011 (PKD), and VA Senior Research Career Scientist Award:1IK6BX005242 (PKD). The studies were also supported by NIH/NIDDK grants, R01 DK54016 and DK92441 (PKD). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of VIPR2 rare and common variants in the Chinese Han population with schizophrenia.

Schizophrenia is a severe and chronic psychiatric disorder with hereditary risk up to 80% as previous studies indicated. Several researches have demonstrated a significant association between schizophrenia and microduplications that overlap the vasoactive intestinal peptide receptor 2 gene (VIPR2). To further investigate potential causal VIPR2 gene variants, all exons and un-translated portions of the VIPR2 gene were sequenced using amplicon targeted resequencing in 1804 Chinese Han patients with schizophrenia and 996 healthy counterparts in the present study. Nineteen rare non-synonymous mutations and 1 frameshift deletion was identified for schizophrenia, among which 5 variants have never been reported so far. Frequencies of rare non-synonymous mutations were significantly different between the two groups. Specifically, the non-synonymous mutation rs78564798 (Pallele = 0.006) as well as two rare variations in the VIPR2 gene's introns (rs372544903, Pallele = 0.026 and a novel mutation, chr7:159034078, GRCh38, Pallele = 0.048) were significantly associated with schizophrenia. Our findings add new evidence that the functional and probable causative variants of VIPR2 gene may play an important role in susceptibility to schizophrenia. Further studies on validations of VIPR2's function in the etiology of schizophrenia are warranted.

VIP and PACAP receptors in GtoPdb v.2023.1

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Vasoactive Intestinal Peptide Receptors [65, 66]) are activated by the endogenous peptides VIP, PACAP-38, PACAP-27, peptide histidine isoleucineamide (PHI), peptide histidine methionineamide (PHM) and peptide histidine valine (PHV). VPAC1 and VPAC2 receptors display comparable affinity for the PACAP peptides, PACAP-27 and PACAP-38, and VIP, whereas PACAP-27 and PACAP-38 are >100 fold more potent than VIP as agonists of most isoforms of the PAC1 receptor. However, one splice variant of the human PAC1 receptor has been reported to respond to PACAP-38, PACAP-27 and VIP with comparable affinity [30]. PG 99-465 [117] has been used as a selective VPAC2 receptor antagonist in a number of physiological studies, but has been reported to have significant activity at VPAC1 and PAC1 receptors [36]. The selective PAC1 receptor agonist maxadilan, was extracted from the salivary glands of sand flies (Lutzomyia longipalpis) and has no sequence homology to VIP or the PACAP peptides [118]. Two deletion variants of maxadilan, M65 [183] and Max.d.4 [119] have been reported to be PAC1 receptor antagonists, but these peptides have not been extensively characterised.

Vasoactive intestinal peptide blockade suppresses tumor growth by regulating macrophage polarization and function in CT26 tumor-bearing mice

Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.

PACAP-PAC1 receptor inhibition is effective in opioid induced hyperalgesia and medication overuse headache models

Opioids prescribed for pain and migraine can produce opioid-induced hyperalgesia (OIH) or medication overuse headache (MOH). We previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) is upregulated in OIH and chronic migraine models. Here we determined if PACAP acts as a bridge between opioids and pain chronification. We tested PACAP-PAC1 receptor inhibition in novel models of opioid-exacerbated trigeminovascular pain. The PAC1 antagonist, M65, reversed chronic allodynia in a model which combines morphine with the migraine trigger, nitroglycerin. Chronic opioids also exacerbated cortical spreading depression, a correlate of migraine aura; and M65 inhibited this augmentation. In situ hybridization showed MOR and PACAP co-expression in trigeminal ganglia, and near complete overlap between MOR and PAC1 in the trigeminal nucleus caudalis and periaqueductal gray. PACAPergic mechanisms appear to facilitate the transition to chronic headache following opioid use, and strategies targeting this system may be particularly beneficial for OIH and MOH.

Vasoactive intestinal peptide receptor 2 signaling promotes breast cancer cell proliferation by enhancing the ERK pathway

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a class B G protein-coupled receptor with the neuropeptide VIP as a ligand. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been documented in several cancers including breast carcinoma. However, the pathophysiological role of increased VIPR2 in the proliferation of breast cancer cells remains largely unknown. In this study, we found that VIPR2 overexpression in MCF-7 and MDA-MB-231 cells, human breast cancer cell lines, promoted cell proliferation. Increased VIPR2 also exacerbated intraperitoneal proliferation of breast cancer MDA-MB-231 cells in a tumor nude mouse model in vivo. Treatment with KS-133, a VIPR2-selective antagonist peptide, significantly inhibited VIP-induced cell proliferation in VIPR2-overexpressing MCF-7 and MDA-MB-231 cells. Overexpressed VIPR2 caused increases in the levels of cAMP and phosphorylated extracellular signal-regulated kinase (ERK), which involves a VIPR2 signaling pathway through Gs protein. Additionally, phosphorylation of vasodilator-stimulated phosphoprotein (Ser157) and cAMP response element binding protein (Ser133) in VIPR2-overexpressing MCF-7 cells was greater than that in control cells, suggesting the increased PKA activity. Moreover, an inhibitor of mitogen-activated protein kinase kinase, U0126, attenuated tumor proliferation in exogenous VIPR2-expressing MCF-7 and MDA-MB-231 cells at the same level as observed in EGFP-expressing cells treated with U0126. Together, these findings suggest that VIPR2 controls breast tumor growth by regulating the cAMP/PKA/ERK signaling pathway, and the excessive expression of VIPR2 may lead to an exacerbation of breast carcinoma.

Secondary bile acid lithocholic acid attenuates neurally evoked ion transport in the rat distal colon.

The inhibitory action of the secondary bile acid lithocholic acid (LCA) on neurally evoked Cl-/HCO3- secretion was investigated using the Ussing-chambered mucosal-submucosal preparation from the rat distal colon. Electrical field stimulation (EFS) evoked cholinergic and noncholinergic secretory responses in the rat distal colon. The responses were almost completely blocked by TTX (10-6 M) but not atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonist for VIP receptor 1 (VPAC1) greatly reduced the EFS-evoked response. Thus, the rat distal colon may be predominantly innervated by noncholinergic VIP secretomotor neurons. Basolateral addition of 6 × 10-5 M LCA inhibited the EFS-evoked response. The inhibitory action of LCA was partly rescued by the Y2R antagonist BIIE0246. The bile acid receptor TGR5 agonist INT-777 mimicked the LCA-induced inhibitory action. Immunohistochemical staining showed the colocalization of TGR5 and PYY on L cells. TGR5 immunoreactivity was also found in VIP-immunoreactive submucosal neurons which also expressed the PYY receptor, Y2R. These results suggest that LCA inhibits neurally evoked Cl-/HCO3- secretion through the activation of TGR5 on L cells and cholinergic- and VIP-secretomotor neurons in the submucosal plexus. Furthermore, the inhibitory mechanism may involve TGR5-stimulated PYY release from L cells and Y2R activation in VIP-secretomotor neurons.

Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer.

Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 μM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 μM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.

Understanding VPAC receptor family peptide binding and selectivity

The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.

Abstract C007: Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

Abstract Current immune checkpoint therapies including anti-CTLA-4 and anti-PD1 have limited efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC), largely due to a paucity of effector T cells within the tumor microenvironment (TME). In our study, we show that vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, is over-expressed in human PDAC tumors. Thus, we hypothesized that paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP-receptor (VIP-R) expressing T cells. We first evaluated whether inhibiting VIP-R signaling by peptide-based VIP-R antagonist affects the growth of PDAC cells in vitro. Treatment of PDAC cells with increasing concentrations of VIP-R antagonist did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 albeit not significant. Furthermore, depletion of VIP receptor 2 (VPAC2) in a PDAC cell line, Panc02, by CRISPR-Cas9 KO had similar growth rates in vitro with a slight growth delay in vivo compared to the parental cell line. These data suggest that the expression of VPAC2 on PDAC cells may not confer a significant autocrine effect on tumor growth impacted by VIP signaling. On the contrary, human T cells cultured with VIP-R antagonists had enhanced activation, decreased proportions of Tregs, and exhausted T cells co-expressing PD-1, Tim-3, and Lag-3. Next, we performed pharmacological inhibition with VIP-R antagonists in tumor-bearing mice to model T cell homing, activation, and antitumor responses in preclinical murine models of PDAC. Daily subcutaneous injections of VIP-R antagonists had synergistic antitumor activity when combined with anti-PD-1 therapy with decreased tumor burden and improved survival. In contrast to the limited single-agent activity of anti-PD1 or VIP-R antagonists, combination therapy eliminated tumors in up to 40% of animals. Anti-tumor responses were T cell-dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. In addition, tumor-free mice were resistant to tumor re-challenge, indicating the generation of anti-cancer immunological memory. In correspondent to these data, combination therapy led to enhanced T cell activation in vivo with increased antigen-specific T cells, decreased frequencies of Tregs as well as enhanced T cell infiltration to the tumor site as observed by infiltration of adoptively transferred GFP+ T cells into PDAC tumors. Taken together, our findings show that VIP-VIPR signaling is a targetable mechanism of immune escape in PDAC. Inhibiting VIP-R signaling improves T cell effector properties and enhances the responsiveness of PDAC to immune checkpoint therapy. Further pharmacokinetic and toxicology studies are underway. Citation Format: Tenzin Passang, Sruthi Ravindranathan, Jian-Ming Li, Shuhua Wang, Michael Brandon Ware, Hanwen Zhang, Gregory B. Lesinski, Edmund K. Waller. Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C007.

High Potency Vasoactive Intestinal Peptide Receptor Antagonists Induce Durable Antileukemia/Tumor T Cell Memory in Mice with Once Daily Dosing

Introduction: Allogeneic bone marrow transplantation (allo-BMT) is curative for patients with myeloid leukemia (AML) through the activity of cytotoxic effector donor T cells, but high morbidity and mortality of allo-BMT limits the use of this therapy. A long-standing goal is the development of strategies that induce an autologous immune response to leukemic blasts by the patient's T cells. Vasoactive intestinal peptide (VIP) is a neuropeptide with pleiotropic functions, including modulation of immune and inflammatory responses. Previous studies demonstrated that VIP hybrid (VIPhyb), a peptide antagonist of VIP-signaling, induced T cell-dependent anti-leukemic activity in murine allotransplant models and promoted activation of mouse and human T cells (Li et al., 2016, Cancer Res). We developed a series of novel peptide VIP antagonists (VIP-ANT) based upon predicted binding affinity in silico to human VIP receptors VPAC1 and VPAC2. We tested the anti-cancer potential of these new VIP-ANT peptides in murine models of myeloid leukemia and mastocytoma, investigating the optimal dose and schedule of administration, and explored mechanisms underlying anti-tumor memory induced by VIP-ANT peptides. Methods: B6 (CD45.1, or albino) mice were injected intravenously with 1 x 106 C1498 cells (AML), and DBA/2j mice were injected subcutaneously with 5 x 104 P815 cells (myeloid mastocytoma). Leukemia-bearing mice were treated with subcutaneous injections of VIP-ANT or control scrambled peptides. Treatment started six days after injection of cells when C1498 leukemia cells were detectable in the blood, and luciferase+ P815 cells as tumors were palpable. A range of VIP-ANT doses was administered s.c. at 10, 30, or 100 μg. Treatment schedules tested included twice daily for 14 doses, once daily, once every other day, once every three days, and once weekly for 7 doses. Some mice received a single dose at day +6 following leukemia cell injection. Lymphocyte kinetics and leukemia cell burden were analyzed weekly through submandibular peripheral blood collections. Mice without evidence of AML/mastocytoma >60 days post-inoculation received a second injection of a two-fold higher dose of C1498 (2 × 106 cells) or P815 (1 × 105 cells). Results: We tested over 13 new VIP-ANT peptides. Daily or every other day (qod) administration of the novel VIP-ANT peptides to leukemia/tumor-bearing mice led to clearance of C1498 cells from blood or complete regression of P815 in 40 % of mice, leading to significantly improved survival over 60 days. Daily administration of 10 μg of novel VIP-ANT peptides for 7 days to leukemic mice prolonged survival(p<0.0001 VIP-ANT vs scramble, Fig 1A), with the fraction of mice that remained leukemia-free proportional to the predicted binding affinity of the VIP-ANT to human VPAC1 and VPAC2. With increased dose intensity and dose-density of ANT308, the most potent peptide, 40%-80% of mice survived to 70 days (median survival days (MSD) of 60 days) after ANT308 treatment compared with the survival of <20% in mice receiving scrambled peptide (MSD 36 days). Increasing the dose and schedule of ANT308 administration to 100 μg twice-daily led to 65% survival (MSD not reached) versus 40% with daily dosing, 30% with every-other-day dosing, and 0% survival with scrambled-sequence control peptide (MSD 33 days) (p<0.001, Fig 1B). Notably, the affinity of different VIP-ANT peptides to VPAC1 and VPAC2 VIP receptors significantly correlated with the survival of C1498-leukemic mice following prior treatment with the corresponding peptide. Prior treatment of leukemic mice with VIP-ANT induced protective immunological memory, rendering mice resistant to re-challenge with C1498 acute myeloid leukemia (80% survival) and P815 mastocytoma (60% survival) cells, compared with 0% survival (MSD 21 days) among immunologically naïve mice. Conclusion: Blocking VIP-signaling with high-affinity VIP-R peptide antagonists represents a novel pharmacological approach to enhance adaptive T cell responses to leukemia/myeloid sarcoma-associated antigens in murine leukemia/tumor models. We are continuing PK and toxicology studies with the lead VIP-ANT peptides to support the development of phase 1 clinical trials for patients with relapsed AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Esophageal mast cells may be associated with the perception of symptoms in patients with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a type 2 helper T-cell (Th2)-mediated allergic disease that involves mast cells. This study aimed to clarify the relationship between perception of symptoms and mast cell levels in patients with EoE. We enrolled patients with asymptomatic esophageal eosinophilia (aEE) and those with symptomatic EoE. Immunofluorescence staining was performed on esophageal biopsy specimens to quantify mast cell-related molecules, such as tryptase, proteinase-activated receptor (PAR)-2, and vasoactive intestinal peptide receptor (VPAC)-1. We evaluated 28 and 58 patients with aEE and EoE, respectively. There were no significant differences in clinical and endoscopic features and peak eosinophil counts between both groups. Mast cell tryptase-positive areas were significantly higher in EoE than in aEE (4.9 [3.5-6.2] vs. 2.0 [1.2-3.4] %, p < 0.01). The number of PAR-2-positive cells was significantly higher in EoE than in aEE (14 [8.8-20.0] vs. 4 [2.8-8.0] cells/high-power field [HPF], p < 0.01). The number of VPAC-1-positive cells was significantly higher in the EoE group than in the aEE group (13 [8.8-16.0] vs. 6 [3.0-9.3] cells/HPF, p < 0.01). A positive correlation was observed between the numbers of PAR-2-positive cells and VPAC-1-positive cells (r = 0.851, p < 0.01). Moreover, mast cell tryptase-positive areas positively correlated with the number of PAR-2- and VPAC-1-positive cells (r = 0.352, p < 0.01; r = 0.355, p < 0.01, respectively). Esophageal mast cells and their receptors, PAR-2 and VPAC-1, may contribute to the perception of symptoms in patients with EoE.