Elsevier

Peptides

Volume 18, Issue 10, 1997, Pages 1539-1545
Peptides

Article
Development of High Affinity Selective VIP1 Receptor Agonists

https://doi.org/10.1016/S0196-9781(97)00228-3Get rights and content

Abstract

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP1 receptor agonists. Peptides 18(10) 1539–1545, 1997.—The biological effects of VIP are mediated by at least two VIP receptors: the VIP1 and the VIP2 receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP1 receptor selective agonists derived from secretin and GRF. [R16]chicken secretin had IC50 values of binding of 1, 10,000, 20, and 3000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP1 receptor (IC50 of 60 nM). The chimeric, substituted peptide [K15,R16,L27]VIP(1-7)/GRF(8-27) had IC50 values of binding of 1, 10,000, 10,000 and 30,000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC50 of 0.8 nM for the human VIP1 receptor and a low affinity for the human VIP2 receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.

Introduction

Vasoactive Intestinal Polypeptide (VIP) is a neuropeptide of 28 amino acids with a widespread distribution in both the central and peripheral nervous system [7]. VIP effects are mediated by high affinity Gs protein coupled receptors.

Molecular cloning revealed the existence of two distinct VIP receptors with seven transmembrane helices named the VIP1 and the VIP2 receptors.

The VIP1 receptor was cloned from rat lung [13]and cancerous human colonic epithelial cells 4, 26; the VIP2 receptor was cloned from the rat olfactory bulb [17]and cerebral cortex, from mouse pancreatic islets [12]and from human SUP T1 lymphoblasts [27]. In situ hybridization to rat organs revealed the expression of VIP1 receptor mRNA [28]in pulmonary large and moderate size bronchi, small intestine, thymus, liver, adrenal medulla, uterine smooth muscle and within the brain in the cerebral cortex and hippocampus; VIP2 receptor mRNA was visualized in mucosa and muscles from the stomach and duodenum, in spleen, thymus, pancreatic islets, the terminal bronchioles, testes [15], ovary, uterus, pituitary, and within the brain, in the thalamus and hypothalamic nuclei. VIP1 receptor mRNA was expressed in human epithelial cell lines [20], rat pituitary cells and tumors [30]and occasionally in human brain tumors [31]and neuroblastomas [32]. VIP2 receptor was also expressed in one glial tumor [33], and in several human cell lines of T origin [20].

VIP1 and VIP2 receptors are pharmacologically distinct: the VIP2 receptor—previously named the helodermin-preferring receptor—had a higher affinity than the VIP1 receptor for helodermin and a lower affinity for secretin and GRF [21]. Several VIP analogues modified in the amino-terminal part of the peptide recognized differently the VIP1 and the VIP2 receptors 9, 22. The affinity or selectivity of these peptides were too low to allow them to be useful tools for receptor classes identification.

We recently discovered [10]that the introduction of an arginine residue in position 16 of VIP or Pituitary Adenylate Cyclase Activating Peptide (PACAP) increased the peptide affinity for the VIP and PACAP receptors and also that the same substitution in secretin reduced its affinity for the secretin receptors. We took advantage of that finding to synthesize derivatives with a high affinity and selectivity for the VIP1 receptors. We focused our attention on that receptor class as a high affinity and highly selective VIP2 agonist was already available [11].

Section snippets

Transfection, Selection and Maintenance of Cell Lines

The CHO cell lines expressing the rat PACAP I receptor [2], the rat VIP1 receptor [3], the human VIP2 receptor [27]and the rat secretin receptor 14, 34have already been described. The CHO cells expressing the recombinant rat VIP2 receptor was kindly provided by Dr. E.M. Lutz from the MRC, Brain Metabolism Unit, Edinburgh. The LoVo cells that express the human VIP1 receptor were cultured as described [11].

Membrane Preparation and Receptor Identification

Transferred CHO cells were harvested with a rubber policeman and pelleted by low speed

Synthesis of a Selective VIP1 Receptor Agonist Derived from Secretin

Secretin has a high affinity for the secretin receptor and a low affinity for the VIP receptors (Table 1 and Fig. 1, Fig. 2). However, it discriminates between the two VIP receptor subclasses, having a clear preference for the VIP1 receptor. This is observed on both rat and human receptors although the human VIP1 receptor had a lower affinity than the rat receptor. Secretin had a negligible affinity for the PACAP type I selective receptor.

The introduction in position 16 of an arginine residue

Discussion

The biological effects of VIP are mediated through interaction with two high affinity receptors, the VIP1- and the VIP2 receptors,[19]that were cloned in rat, human, and mice tissues 4, 12, 13, 17, 26, 27.

The mRNA coding for each receptor is distributed differently in brain and peripheral tissues 15, 28suggesting that receptor occupancy may lead to different activity profiles. To test this hypothesis and define these properties it is necessary to use selective agonist—or antagonists—for each

Acknowledgements

Aided by Grant no 3.4502.95 from the Fonds de la Recherche Scientifique Médicale, Belgium and by an Action de Recherche Concertée from the “Communauté Française de Belgique” and by a “Interuniversity Poles of Attraction Programme—Belgian State, Prime Minister’s Office—Federal Office for Scientific, Technical and Cultural Affairs.”

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