Selective VPAC2 Research Articles

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.

Expression and function of vasoactive intestinal peptide receptors in human lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is an important neurotransmitter involved in the modulation of gastrointestinal function through the stimulation of VIP receptors. However, the expression of VPAC1R, VPAC2R and PAC1R in the human Lower esophageal sphincter (LES) has not been fully clarified. Therefore, the purpose of this study is to explore the expression of these receptors in the human Lower esophageal sphincter, the responses of the Lower esophageal sphincter to Vasoactive intestinal peptide, and the role of Vasoactive intestinal peptide receptors in the responses. Sling and clasp fiber samples of LES were acquired from patients undergoing subtotal esophagectomy, while circular muscle bundles from the esophagus and gastric fundus were used as control groups. Western blotting and RT-PCR technology were performed to determine the expression of the three VIP receptor subtypes. The isometric tension responses of the muscle sample strips to Ro25-1553 and PG99-465, and the effect of electrical field stimulation (EFS) on the sling and clasp fibers were studied. We found that VPAC2R messenger RNA (mRNA) and protein were expressed in the sling and clasp fibers of human LES. However, no VPAC1R or PAC1R mRNA and protein expressions were found in the LES samples. The sling and clasp fibers of the LES produced significant concentration-dependent relaxation following exposure to Ro25-1553 and EFS could induce them to produce frequency-dependent relaxation. Furthermore, the relaxation responses of the LES were inhibited by PG99-465 and induced by EFS and Ro25-1553. VPAC2R, but not VPAC1R or PAC1R, is expressed by the human LES. The relaxation responses of the LES generated by the VIP receptor agonist Ro25-1553 and EFS could be inhibited by the selective VPAC2 receptor antagonist PG99-465. VPAC2R may be important for the generation of relaxation and functional regulation of the LES.

Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism.

Clinical studies have shown that microduplications at 7q36.3, containing VIPR2, confer significant risk for schizophrenia and autism spectrum disorder (ASD). VIPR2 gene encodes the VPAC2 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP-induced cAMP responsiveness, but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We have previously found that repeated administration of a selective VPAC2 receptor agonist Ro25-1553 in the mouse during early postnatal development caused synaptic alterations in the prefrontal cortex and sensorimotor gating deficits. In this study, we aimed to clarify the effects of VPAC2 receptor activation on neurite outgrowth in cultured primary mouse cortical neurons. Ro25-1553 and VIP caused reductions in total numbers and lengths of both neuronal dendrites and axons, while PACAP38 facilitated elongation of dendrites, but not axons. These effects of Ro25-1553 and VIP were blocked by a VPAC2 receptor antagonist PG99-465 and abolished in VPAC2 receptor-deficient mice. Additionally, Ro25-1553-induced decreases in axon and dendritic outgrowth in wild-type mice were blocked by a protein kinase A (PKA) inhibitor H89, but not by a PKC inhibitor GF109203X or a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor U0126. PACAP38- induced facilitation of dendritic outgrowth was blocked by U0126. These results suggest that activation of the VPAC2 receptor impairs neurite outgrowth and decreases branching of cortical neurons by a PKA-dependent mechanism. These findings also imply that the VIPR2-linkage to mental health disorders may be due in part to deficits in neuronal maturation induced by VPAC2 receptor overactivation.

VIP and PACAP receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

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 [64, 65]) 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 [29]. PG 99-465 [115] 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 [35]. 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 [116]. Two deletion variants of maxadilan, M65 [180] and Max.d.4 [117] have been reported to be PAC1 receptor antagonists, but these peptides have not been extensively characterised.

A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects.

A novel neuroendocrine peptide, pituitary adenylate cyclase activating peptide (PACAP), was found to have an important role in carbohydrate or lipid metabolism and was susceptible to dipeptidyl peptidase IV degradation. It can not only mediate glucose-dependent insulin secretion and lower blood glucose by activating VPAC2 receptor, but also raise blood glucose by promoting glucagon production by VPAC1 receptor activation. Therefore, its therapeutic application is restricted by the exceedingly short-acting half-life and the stimulatory function for glycogenolysis. Herein, we generated novel peptide-conjugated selenium nanoparticles (SeNPs; named as SCD), comprising a 32-amino acid PACAP-derived peptide DBAYL that selectively binds to VPAC2, and chitosan-modified SeNPs (SeNPs-CTS, SC) as slow-release carrier. The circulating half-life of SCD is 14.12 h in mice, which is 168.4-and 7.1-fold longer than wild PACAP (~5 min) and DBAYL (~1.98 h), respectively. SCD (10 nmol/L) significantly promotes INS-1 cell proliferation, glucose uptake, insulin secretion, insulin receptor expression and also obviously reduces intracellular reactive oxygen species levels in H2O2-injured INS-1 cells. Furthermore, the biological effects of SCD are stronger than Exendin-4 (a clinically approved drug through its insulinotropic effect), DBAYL, SeNPs or SC. A single injection of SCD (20 nmol/kg) into db/db mice with type 2 diabetes leads to enhanced insulin secretion and sustained hypoglycemic effect, and the effectiveness and duration of SCD in enhancing insulin secretion and reducing blood glucose levels are much stronger than Exendin-4, SeNPs or SC. In db/db mice, chronic administration of SCD by daily injection for 12 weeks markedly improved glucose and lipid profiles, insulin sensitivity and the structures of pancreatic and adipose tissue. The results indicate that SC can play a role as a carrier for the slow release of bioactive peptides and SCD could be a hopeful therapeutic against type 2 diabetes through the synergy effects of DBAYL and SeNPs.

Reductions in synaptic proteins and selective alteration of prepulse inhibition in male C57BL/6 mice after postnatal administration of a VIP receptor (VIPR2) agonist.

An abundance of genetic and epidemiologic evidence as well as longitudinal neuroimaging data point to developmental origins for schizophrenia and other mental health disorders. Recent clinical studies indicate that microduplications of VIPR2, encoding the vasoactive intestinal peptide (VIP) receptor VPAC2, confer significant risk for schizophrenia and autism spectrum disorder. Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP responsiveness (cAMP induction), but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We subcutaneously administered the highly selective VPAC2 receptor agonist Ro 25-1553 to C57BL/6 mice from postnatal day 1 (P1) to P14 to determine if overactivation of VPAC2 receptor signaling during postnatal brain maturation affects synaptogenesis and selected behaviors. Western blot analyses on P21 revealed significant reductions of synaptophysin and postsynaptic density protein 95 (PSD-95) in the prefrontal cortex, but not in the hippocampus in Ro 25-1553-treated mice. The same postnatally restricted treatment resulted in a disruption in prepulse inhibition of the acoustic startle measured in adult mice. No effects were observed in open-field locomotor activity, sociability in the three-chamber social interaction test, or fear conditioning or extinction. Overactivation of the VPAC2 receptor in the postnatal mouse results in a reduction in synaptic proteins in the prefrontal cortex and selective alterations in prepulse inhibition. These findings suggest that the VIPR2-linkage to mental health disorders may be due in part to overactive VPAC2 receptor signaling during a critical time of synaptic maturation.

Endogenous inhibition of hippocampal LTD and depotentiation by vasoactive intestinal peptide VPAC1 receptors.

Vasoactive intestinal peptide (VIP), an important modulator of hippocampal synaptic transmission, influences exploration and hippocampal-dependent learning in rodents. Homosynaptic long-term depression (LTD) and depotentiation are two plasticity phenomena implicated in learning of behavior flexibility and spatial novelty detection. In this study, we investigated the influence of endogenous VIP on LTD and depotentiation induced by low-frequency stimulation (1 Hz, 900 pulses) of the hippocampal CA1 area in vitro in juvenile and young adult rats, respectively. LTD and depotentiation were enhanced by the VIP receptor antagonist Ac-Tyr(1) , D-Phe(2) GRF (1-29), and the selective VPAC1 receptor antagonist, PG 97-269, but not the selective VPAC2 receptor antagonist, PG 99-465. This action was mimicked by an anti-VIP antibody, suggesting that VIP, and not pituitary adenylate cyclase-activating polypeptide (PACAP), is the endogenous mediator of these effects. Selective inhibition of PAC1 receptors with PACAP (6-38) enhanced depotentiation, but not LTD. VPAC1 receptor blockade also revealed LTD in young adult rats, an effect abolished by the GABAA antagonist bicuculline, evidencing an involvement of GABAergic transmission. We conclude that inhibition of LTD and depotentiation by endogenous VIP occurs through VPAC1 receptor-mediated mechanisms and suggest that disinhibition of pyramidal cell dendrites is the most likely physiological mechanism underlying this effect. As such, VPAC1 receptor ligands may be considered promising pharmacological targets for treatment of cognitive dysfunction in diseases involving altered GABAergic circuits and pathological saturation of LTP/LTD like Down's syndrome and temporal lobe epilepsy.

Activation of the VIP/VPAC2 system induces reactive astrocytosis associated with increased expression of glutamate transporters

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide that acts as a neuromodulator in the CNS. Recently, secretion of several functional molecules has been identified in VIP-stimulated astrocytes in vitro. However, the relationship between VIP and its specific receptors in neurological disorders remains unknown. To investigate the role of the VIP system under pathological conditions, we performed a cold injury on the right cerebrum of adult C57BL/6 mice and observed expression patterns for VIP and its receptor, VPAC2. Immunohistochemical studies revealed VPAC2 expression in reactive astrocytes around the core lesion by post-injury day 7, which then returned to contralateral levels at post-injury day 14. By contrast, VIP immunoreactivity was detected in activated microglial cells, suggesting that microglia–astrocyte interactions in the VIP/VPAC2 system are important for the tissue repair process. In primary cultured astrocytes stimulated with N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP) to mimic reactive astrocytosis, VPAC2 mRNA expression was highly up-regulated compared to that of the other VIP receptors, PAC1 and VPAC1. VPAC2 activation by the selective VPAC2 agonist, Ro25-1553, induced reactive morphological and biochemical changes from a polygonal shape to a stellate shape in cultured astrocytes. Further, Ro25-1553 increased cell surface expression of the glutamate transporters GLAST and GLT-1, which can limit excitotoxic neuronal cell death. In summary, the transient expression of VPAC2 in reactive astrocytes and the up-regulation of functional glutamate transporters suggest that the VIP/VPAC2 system induces reactive astrocytosis and plays a key role in neuroprotection against excitotoxicity in neurological disorders.

Dual bronchodilatory and pulmonary anti‐inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors

Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC(1) and VPAC(2). RO5024118 is a selective VPAC(2) receptor agonist derived via chemical modification of an earlier VPAC(2) agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118. Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model. RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting β-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting β-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8(+) cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation. These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases. This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Effects of VPAC2 Receptor Activation on Membrane Excitability and GABAergic Transmission in Subparaventricular Zone Neurons Targeted by Suprachiasmatic Nucleus

The hypothalamic suprachiasmatic nucleus (SCN) harbors the master circadian pacemaker. SCN neurons produce the amino acid gamma-aminobutyric acid (GABA) and several peptide molecules for coordination and communication of their circadian rhythms. A subpopulation of SCN cells synthesizes vasoactive intestinal polypeptide (VIP) and provides a dense innervation of the subparaventricular zone (SPZ), an important CNS target of the circadian pacemaker. In this study, using patch-clamp recording techniques and rat brain slice preparations, the contribution of VIP to SCN efferent signaling to SPZ was evaluated by examining membrane responses of SPZ neurons to exogenous VIP receptor ligands. In approximately 50% of the SPZ neurons receiving monosynaptic GABAA receptor-mediated inputs from SCN, bath-applied VIP (0.5-1 microM) resulted in a membrane depolarization caused by tetrodotoxin-resistant inward currents reversing at approximately -23 mV. These data suggest the existence of postsynaptic receptors that activate a nonselective cationic conductance. In addition, a subset of SPZ neurons showed an increase in the amplitude of SCN-evoked GABAergic inhibitory postsynaptic currents (IPSCs) and a decrease in their paired-pulse ratios. This, together with an increase in frequency of spontaneous and miniature IPSCs, implies the presence of presynaptic receptors that facilitate GABA release from SCN and possibly other synaptic terminals. The effects occurred in separate neurons and could be mimicked by the selective VPAC2 receptor agonist BAY 55-9837 (0.2-0.5 microM) and partially blocked by the VIP receptor antagonist VIP(6-28) (5 microM). The results indicate that VIP acts via both post- and presynaptic VPAC2 receptors to differentially modulate SCN GABAergic signaling to distinct subpopulations of SPZ neurons.

Mechanisms of vasoactive intestinal peptide-elicited coronary vasodilation in the isolated perfused rat heart

The present study investigated the potential role of vasoactive intestinal peptide (VIP) receptors, VPAC1 and VPAC2, in VIP-elicited coronary vasodilation of the isolated perfused rat heart. Additional studies determined the role of ATP-sensitive (K(ATP)) and voltage-gated K(+) (K(V)) channels in the VIP-elicited coronary vasodilation. Both the selective VPAC1 agonist, K15,R16,L27VIPl-7GRF8-27, and the selective VPAC2 agonist, RO25-1553, decreased coronary vascular resistance (CVR) in a dose-dependent manner, with EC(50) values of 1.67x10(-9)M and 7.11x10(-9)M, respectively (VPAC1 vs VPAC2 agonist, P<0.05). K15,R16,L27VIP1-7GRF8-27 and RO25-1553 maximally reduced CVR by -42+/-4% and -39+/-6% at 1x10(-8) and 3x10(-8)M, respectively. VIP at 1x10(-10)M decreased CVR by -14+/-2% in the absence (vehicle), by -11+/-3% in the presence of the nonselective VIP receptor antagonist VIP10-28 (1x10(-7)M; P>0.05 vs. vehicle) and by only -4+/-2% in the presence of the selective VPAC2 receptor antagonist PACAP6-38 (1x10(-7)M; P<0.05 vs. vehicle). In additional studies, VIP at 1x10(-10)M decreased CVR by -22+/-1% in the absence (control) and by only -10+/-2% in the presence of the nonselective K(+) channel blocker tetrabutylammonium (3x10(-4)M; P<0.05 vs. control). VIP reduced CVR by -4+/-1% in the presence of the K(ATP) channel blocker glibenclamide (3x10(-6)M; P<0.05 vs control) and by -28+/-2% in the presence of the K(V) channel blocker 4-aminopyridine (3x10(-4)M; P>0.05 vs control). Thus, selective VPAC1 and VPAC2 receptor activation in the coronary circulation produces vasodilation and the VIP-elicited coronary vasodilation involves activation of VPAC2 receptors and K(ATP) but not K(V) channels. In addition, VIP10-28 does not effectively block coronary vascular VIP receptors.

Neurogenic secretion mediated by the purinergic P2Y 1 receptor in guinea-pig small intestine

We tested the hypothesis that ATP is an enteric neurotransmitter that acts at P2Y 1 excitatory purinergic receptors on intestinal secretomotor neurons to evoke neurogenic mucosal secretion in the guinea pig. Ussing chamber methods for studying neurogenic intestinal secretion were used to test the hypothesis. Application of ATP evoked concentration-dependent increases in short circuit current ( Isc) indicative of stimulation of electrolyte secretion. MRS2179, a selective P2Y 1 purinergic receptor antagonist, suppressed the ATP-evoked responses in a concentration-dependent manner with an IC 50 of 0.9 ± 0.1 μM. Tetrodotoxin or a selective vasoactive intestinal peptide (VPAC 1) receptor antagonist suppressed or abolished the ATP-evoked responses. A selective VPAC 1 receptor antagonist also suppressed Isc responses evoked by electrical field stimulation of the secretomotor neurons. Secretory responses to ATP were not suppressed by scopolamine, piroxicam nor selective adenosine receptor antagonists. Region-specific differences in responses to ATP corresponded to regional differences in the expression of mRNA transcripts for the P2Y 1 receptor. Post-receptor signal transduction for the P2Y 1-evoked responses involved stimulation of phospholipase C and an IP3/Ca 2+-calmodulin/protein kinase C signaling cascade. Our evidence suggests that ATP is released as a neurotransmitter to stimulate neurogenic mucosal secretion by binding to P2Y 1 receptors expressed by VIP-ergic secretomotor neurons.

Ac His 1 [ d-Phe 2, K 15, R 16, L 27] VIP (3–7)/GRF (8–27) – a VPAC 1 receptor antagonist – is an inverse agonist on two constitutively active truncated VPAC 1 receptors

C-terminally truncated human VPAC 1 receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand’s binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1–457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q 3-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G αs protein. Cells expressing the VPAC 1 (1–436) and (1–441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC 1 (1–444), (1–433), (1–429), (1–421) and (1–398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC 1 (1–436) and (1–441) receptors had a constitutive activity. The selective VPAC 1 receptor antagonist Ac His 1 [ d-Phe 2, K 15, R 16, L 27] VIP (3–7)/GRF (8–27) reduced by 60% the basal activity with an EC 50 value of 3 nM comparable to its IC 50 value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC 1 receptors generated by C-terminal truncation and expressed in CHO cells.

Distribution of the VPAC2 Receptor in Peripheral Tissues of the Mouse

The neuropeptide vasoactive intestinal peptide (VIP) exerts its actions through two structurally related G protein-coupled receptors (VPAC(1) and VPAC(2)). Pituitary adenylate cyclase-activating polypeptide (PACAP) is also a potent agonist of VPAC(1) and VPAC(2) receptors as well as of a third, PACAP-specific receptor (PAC(1)). We report here the distribution of the VPAC(2) receptor in peripheral tissues of the mouse, determined by receptor autoradiography using [(125)I]VIP and the selective VPAC(2) receptor agonist [(125)I]Ro25-1553 in wild-type and VPAC(2) receptor-null mice. In addition, displacement experiments with the VPAC(2)-selective agonist Ro25-1553 and the VPAC(1)-selective agonist [K(15),R(16),L(27)]VIP(1-7)/GRF(8-27) were performed using the universal radioligand [(125)I]VIP. The VPAC(2) receptor is found predominantly in smooth muscle (in blood vessels and in the smooth muscle layers of the gastrointestinal and reproductive systems), the basal part of the mucosal epithelium in the colon, lung, the vasculature of the kidney, adrenal medulla, and retina. Unexpectedly, the receptor was also present in thyroid follicular cells and acinar cells of the pancreas, tissues that have not been found to express the receptor in other species, and in very large amounts in the lung. Our data suggest novel functions of the VPAC(2) receptor and additional potential therapeutic uses of drugs acting at the receptor (including the treatment of erectile dysfunction), but our results also indicate that caution should be exercised in using the mouse as an animal model for the evaluation of VIP analogs intended for diagnostic or therapeutic use in man.

Regulation of inhibitory synaptic transmission by vasoactive intestinal peptide (VIP) in the mouse suprachiasmatic nucleus.

Circadian rhythmicity in mammals is generated by a pair of nuclei in the anterior hypothalamus known as the suprachiasmatic nuclei (SCN), whose neurons express a variety of neuropeptides that are thought to play an important role in the circadian timing system. To evaluate the influence of VIP on inhibitory synaptic transmission between SCN neurons, we used whole cell patch-clamp recording in an acute brain slice preparation of mouse SCN. Baseline spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) varied significantly between regions and across phases, with a greater frequency of IPSCs observed in the dorsomedial region during the early night. Bath-applied VIP caused a significant increase in the frequency of spontaneous inhibitory postsynaptic currents (sIPSC) in a reversible and dose-dependent manner with no effect on the mean amplitude or kinetic parameters. The effect of VIP was widespread throughout the SCN and observed in both ventrolateral (VL) and dorsomedial (DM) regions. In the presence of tetrodotoxin, VIP increased the frequency of miniature IPSCs without affecting the mean magnitude or kinetic parameters. The magnitude of the enhancement by VIP was significantly larger during the day than during the night. Pretreatment with the VIP-PACAP receptor antagonist [Ac-Tyr1, D-Phe2]-GHRF 1-29 or the selective VPAC2 receptor antagonist PG 99-465 completely blocked the VIP-induced enhancement. The effect of VIP appears to be mediated by a cAMP/PKA-dependent mechanism as forskolin mimics, while the PKA antagonist H-89 blocks the observed enhancement of GABA currents. Our data suggest that VIP activates presynaptic VPAC2 receptors to regulate inhibitory synaptic transmission within the SCN and that this effect varies from day to night.

Generation of Highly Selective VPAC2 Receptor Agonists by High Throughput Mutagenesis of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-activating Peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.

Effects of vasoactive intestinal polypeptide on neurones of the rat suprachiasmatic nuclei in vitro.

The suprachiasmatic nuclei (SCN) of the hypothalamus house the main circadian pacemaker in mammals. Vasoactive intestinal polypeptide (VIP) is the most abundant neuropeptide in the SCN and has been shown to phase-shift the electrical activity rhythm of SCN cells in vitro. However, the effects of VIP on the cellular activity of rat SCN neurones are unknown. In this study, we examined the acute effects of VIP on the extracellularly recorded spontaneous firing rate of SCN neurones in an in-vitro hypothalamic slice preparation. Furthermore, with the use of receptor-selective agonists and antagonists, we determined which receptors might mediate the effects of VIP in the SCN. Approximately 50% of cells responded to VIP; the main type of response was suppression in firing rate, although a few cells were activated. Suppression responses to VIP were mimicked by the VPAC(2) receptor agonist Ro 25-1553 and blocked by the selective VPAC(2) receptor antagonist PG 99-465. The PAC(1) receptor agonist maxadilan evoked responses from 40% of SCN cells, and activations to this agonist were not altered by PG 99-465. Responses to VIP were not blocked by antagonists to ionotropic glutamate receptors, but the duration of suppression was modulated by the GABA(A) receptor antagonist bicuculline. Our data indicate that VIP alters the electrical activity of rat SCN neurones in vitro, via both VPAC(2) and PAC(1) receptors.

A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes.

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K(d)) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.

Vasoactive intestinal polypeptide (VIP) phase-shifts the rat suprachiasmatic nucleus clock in vitro.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC(2) and PAC(1), are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase-resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase-advancing effect of VIP was reproduced by the novel VPAC(2) receptor agonist RO 25-1553, but not by pituitary adenylate cyclase-activating peptide (a potent PAC(1) receptor agonist), or by [K15,R16,L27]VIP(1-7)/GRF(8-27), a novel, selective VPAC(1) receptor agonist. These data show that VIP phase-dependently phase-resets the rodent SCN pacemaker in vitro, presumably via the VPAC(2) receptor. As the pattern of phase-shifting evoked by VIP and RO 25-1553 resembles the phase-resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC(2) receptor in photic entrainment of the rodent circadian pacemaker.

Creation of a selective antagonist and agonist of the rat VPAC(1) receptor using a combinatorial approach with vasoactive intestinal peptide 6-23 as template.

We have used combinatorial chemistry with amino acid mixtures (X) at positions 6 to 23 in vasoactive intestinal peptide (VIP) to optimize binding affinity and selectivity to the rat VPAC(1) receptor. The most efficient amino acid replacement was a substitution of alanine at position 18 to diphenylalanine (Dip), increasing the displacement efficiency of (125)I-VIP by 370-fold. The [Dip(18)]VIP(6-23) was subsequently used to find a second replacement, employing the same approach. Tyrosine at position 9 was selected and the resulting [Tyr(9),Dip(18)]VIP(6-23) analog has a K(i) value of 90 nM. This analog was unable to stimulate cAMP production at 10(-6) M but was able to inhibit VIP-induced cAMP stimulation (K(b) = 79 nM). The K(i) values of [Tyr(9),Dip(18)]VIP(6-23) using the rat VPAC(2) and PAC(1) receptors were 3,000 nM and >10,000 nM, respectively. Thus, [Tyr(9),Dip(18)]VIP(6-23) is a selective VPAC(1) receptor antagonist. The C-terminally extended form, [Tyr(9),Dip(18)]VIP(6-28), displays improved antagonistic properties having a K(i) and K(b) values of 18 nM and 16 nM, respectively. On the contrary, the fully extended form, [Tyr(9),Dip(18)]VIP(1-28), was a potent agonist with improved binding affinity (K(i) = 0.11 nM) and ability to stimulate cAMP (EC(50) = 0.23 nM) compared with VIP (K(i) = 1.7 nM, EC(50) = 1.12 nM). Furthermore, the specificity of this agonist to the VPAC(1) receptor was high, the K(i) values for the VPAC(2) and PAC(1) receptors were 53 nM and 3,100 nM, respectively. Seven other analogs with the [Tyr(9),Dip(18)] replacement combined with previously published VIP modifications have been synthesized and described in this work.