The Long-Acting Vasoactive Intestinal Polypeptide Agonist RO 25-1553 Is Highly Selective of the VIP2 Receptor Subclass

doi:10.1016/S0196-9781(96)00322-1

Peptides

Volume 18, Issue 3, 1997, Pages 403-408

https://doi.org/10.1016/S0196-9781(96)00322-1 Get rights and content

Abstract

Gourlet, P., P. Vertongen, A. Vandermeers, M.-C. Vandermeers-Piret, J. Rathe, P. De Neef, M. Waelbroeck and P. Robberecht. The long-acting vasoactive intestinal polypeptide agonist RO 25-1553 is highly selective of the VIP₂ receptor subclass. Peptides 18(3) 403–408, 1997.—RO 25-1553 is a synthetic VIP analogue that induced a long-lasting relaxation of tracheal and bronchial smooth muscles as well as a reduction of edema and eosinophilic mobilization during pulmonary anaphylaxis. In the present study, we tested in vitro the capacity of RO 25-1553 to occupy the different VIP/PACAP receptor subclasses and to stimulate adenylate cyclase activity. The cellular models tested expressed one single receptor subtype: Chinese hamster ovary (CHO) cells transfected with the rat recombinant PACAP I, rat VIP₁, and human VIP₂ receptors; SUP T1 cells expressing the human VIP₂ and HCT 15 and LoVo cells expressing the human VIP₁ receptor. RO 25-1553 was threefold more potent than VIP on the human VIP₂ receptor, 100- and 600-fold less potent than VIP on the rat and human VIP₁ receptors, respectively, and 10-fold less potent than VIP and 3000-fold less potent than PACAP on the PACAP I receptor. RO 25-1553 was a full agonist on the VIP₂, the PACAP I, and the rat recombinant VIP₁ receptor but a partial agonist only on the human VIP₁ receptor. Thus, RO 25-1553 is a highly selective agonist ligand for the VIP₂ receptor subclass.

Section snippets

Peptide Synthesis

VIP, PACAP-27, and PACAP-38 were synthesized by solid-phase methodology using the Fmoc strategy. RO 25-1553 was synthesized as described [19]using the N^α-Boc derivatives. All the peptides were purified on various HPLC and FPLC columns. The purity of the material was at least 95% as judged by capillary electrophoresis and analytical reverse-phase chromatography and the peptide conformity was assessed by mass spectrometry.

Cell Cultures

Chinese hamster ovary cells (CHO cells) expressing the recombinant rat VIP₁

Characteristics of the Cell Lines

The characteristics of the CHO cell lines transfected with the DNA coding for the rat VIP₁, human VIP₂, and rat PACAP type I receptors were the following: [¹²⁵I]VIP recognized 850 ± 50 and 100 ± 30 fmol of rat VIP₁ receptor/mg protein on VIP₁ r clone 3 and 16 cells, respectively [6]; 210 ± 40 fmol of human VIP₂ receptor/mg protein on the VIP₂ r clone 11 cells [27], and [¹²⁵I]PACAP-27 detected 4.6 ± 0.3 pmol of rat PACAP I receptor/mg protein on PACAP I r clone P2-10 cells [7]. mRNA was prepared

Discussion

VIP/PACAP receptors are classified as PACAP type I receptors with a high affinity for PACAP and a low affinity for VIP, and PACAP type II receptors, with an equal high affinity for PACAP and VIP. The PACAP type II receptors were further subdivided into VIP₁ and VIP₂ receptors 5, 21. VIP₁ receptors have been cloned in rat [13]and human [8]; VIP₂ receptors have been cloned in rat [17], mouse [12], and human [27]. The VIP₂ receptor corresponds to the “helodermin-preferring” receptor previously

Acknowledgements

Aided by Grant No. 3.4502.95 from the Fonds de la Recherche Scientifique Médicale and by an Action Concertée de Recherche from the Communauté Française de Belgique.

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Although this study was primarily aimed at identifying VIP/PACAP-analogs with selectivity for PAC1 over VPAC1, their selectivity for PAC1 over VPAC2 was also examined (Table 5). Whereas, a number of selective VPAC1-R agonists ([Ala11,22,28]VIP, [Ala2,8,9,16,19,24]VIP and [Lys15,Arg16,Leu27]VIP(1–7)/GHRH(8–27)) and selective VAC2 agonists (Ro 25-1553 and Ro 25-1392) have been described [11,17,19,24,40,60], a few agonists selective for PAC1-R/VPAC1-R over VPAC2-R have also been described [13,16]. There are also a number of structure–function studies comparing VPAC1-R and VPAC2-R that have provided important insights into VIP analogs that can discriminate these two closely related receptors [11,23–25,40,41,55].
Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.
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Leptin and resistin are adipokines considered as pro-inflammatory factors related to metabolic syndrome, inflammatory and/or autoimmune conditions. Pituitary adenylate cyclase activating peptide (PACAP) is a pleiotropic neuropeptide with anti-inflammatory properties. We investigated the influence of PACAP on the serum level of leptin, soluble leptin receptor (SLR) and resistin in ordinary and LPS-induced inflammatory conditions using PACAP38 and a series of selective agonist for each PACAP receptor types. It was found that PACAP exerted opposite effects on the leptin:SLR ratio and the serum resistin level. In ordinary condition, PACAP acted as a pro-inflammatory factor by increasing the leptin:SLR ratio and serum resistin level. But in LPS-induced acute inflammatory condition, PACAP not only antagonized the effects of LPS, but also even reversed the effects of LPS. In mice treated with LPS, co-treatment with PACAP decreased the serum leptin and resistin levels and increased the serum soluble leptin receptor level significantly. It was also found that, in ordinary condition, treatment with PAC1 agonist maxadilan induced marked increase in serum leptin, leptin:SLR ratios and resistin levels; while in LPS-induced inflammation, VPAC1 mediated much more anti-inflammatory and reversing-LPS effects of PACAP on leptin and resistin than PAC1 and VPAC2. It is concluded that different receptors mediates different effects of PACAP on leptin, SLR and resistin in non-inflammatory and LPS-induced inflammatory conditions.
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Citation Excerpt :
The majority of cell based studies examining endogenously expressed hVPAC2R receptors have focused on SUP-T1 cells, however some have used Molt-4b lymphoblastic leukaemia cells (Summers et al., 2003) and THP-1 monocytic leukaemia cells (Chedeville et al., 1993). From the SUP-T1 studies, the generally observed ROP is helodermin > VIP = PACAP = PHI > > secretin, although a degree of variability is evident between studies (Robberecht et al., 1988, 1989; Xia et al., 1996; Gourlet et al., 1997c; Igarashi et al., 2002). Average EC50 values reported in these studies from the examination of cAMP production, were ~ 0.3 nM for helodermin and ~ 10 nM for VIP and the PACAPs.
Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC₁, VPAC₂ and PAC₁R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.
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Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.
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The Long-Acting Vasoactive Intestinal Polypeptide Agonist RO 25-1553 Is Highly Selective of the VIP2 Receptor Subclass

Abstract

Section snippets

Peptide Synthesis

Cell Cultures

Characteristics of the Cell Lines

Discussion

Acknowledgements

FEBS Lett.

Biochim. Biophys. Acta

Regul. Pept.

Eur. J. Pharmacol.

Biochem. Biophys. Res. Commun.

Regul. Pept.

Eur. J. Pharmacol.

Neuron

Neuropeptides

Peptides

FEBS Lett.

J. Biol. Chem.

Mol. Cell. Endocrinol.

FEBS Lett.

Adv. Neuroimmunol.

The Long-Acting Vasoactive Intestinal Polypeptide Agonist RO 25-1553 Is Highly Selective of the VIP₂ Receptor Subclass