ArticleDevelopment of High Affinity Selective VIP1 Receptor Agonists
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].
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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.”
References (36)
- et al.
Pharmacological properties of two recombinant splice variants of the PACAP type I receptor transfected and stably expressed in CHO cells
Eur. J. Pharmacol.
(1995) - et al.
Properties of the VIP-PACAP type II receptor stably expressed in CHO cells
Regul. Pept.
(1994) - et al.
Human intestinal VIP receptorCloning and functional expression of two cDNA encoding proteins with different N-terminal domains
Biochem. Biophys. Res. Commun.
(1994) - et al.
Amino acid sequence of VIP, PHI and secretin from the rabbit small intestine
Peptides
(1990) - et al.
The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes
Biochim. Biophys. Acta
(1991) - et al.
Effect of introduction of an Arginine16 in VIP, PACAP and secretin on ligand affinity of the receptors
Biochim. Biophys. Acta
(1996) - et al.
; and Robberecht, P. The long-acting vasoactive intestinal polypeptide agonist RO 25–1553 is highly selective of the VIP2 receptor subclass
Peptides
(1997) - et al.
Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide
Neuron
(1992) - et al.
PACAP acts through VIP type 2 receptors in the rat testis
Neuropeptides
(1995) - et al.
Interaction of GRF with VIP receptors and stimulation of adenylate cyclase in rat and human intestinal epithelial membranes
Comparison with PHI and secretin. FEBS Lett.
(1983)
The VIP2 receptormolecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide
FEBS Lett.
Isolation, aminoacid composition and terminal amino acid residues of the vasoactive octacosapeptide from chicken intestine
Partial purification of chicken secretin. FEBS Lett.
Characterization of the VIP receptor from SUP Tl lymphoblasts
Advan. Neuroimmunol.
A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts
FEBS Lett.
Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes
Regul. Pept.
A highly sensitive adenylate cyclase assay
Anal. Biochem.
Cloning and functional expression of a human neuroendocrine vasoactive intestinal peptide receptor
Biochem. Biophys. Res. Commun.
Molecular cloning and functional characterization of a human VIP receptor from SUP-T1 lymphoblasts
Biochem. Biophys. Res. Commun.
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