André Vandermeers

Development of High Affinity Selective VIP1 Receptor Agonists

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 agonsits 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-, secretion- 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.

The long-acting vasoactive intestinal polypeptide agonist RO 25-1553 is highly selective of the VIP2 receptor subclass

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.

Primary structure of helodermin, a VIP-secretin-like peptide isolated from Gila monster venom

The complete amino acid sequence of helodermin isolated from the venom of Gila monster was elucidated. The peptide was shown to be a basic pentatriacontapeptide amide: His‐Ser‐Asp‐Ala‐Ile‐Phe‐Thr‐Gln‐Gln‐ Tyr‐Ser‐Lys‐Leu‐Leu‐Ala‐Lys‐Leu‐Ala‐Leu‐Gln‐Lys‐Tyr‐Leu‐Ala‐Ser‐Ile‐Leu‐Gly‐Ser‐Arg‐Thr‐Ser‐Pro‐Pro‐Pro‐NH2. A high degree of sequence similarities to secretin/VIP/PHI/(PHM)/GRF from mammal and bird was observed over the entire N‐terminal 1–27 sequence. In particular, the amino acid residues in positions 3, 6 and 7 were found to be common to 9 peptides of the family. Another interesting feature of the structure of helodermin was its C‐terminal ‐Pro‐Pro‐Pro‐NH2 sequence. Isolation of helodermin was the first demonstration of the existence of a secretin/VIP‐related peptide in an animal that is neither mammal nor bird.

Properties and distribution of receptors for pituitary adenylate cyclase activating peptide (PACAP) in rat brain and spinal cord

A high density (in the pmol/mg protein range) of specific functional receptors for PACAP (pituitary adenylate cyclase activating polypeptide) was observed in membranes from rat brain cortex, olfactory bulb, hypothalamus, hippocampus, striatum, cerebellum, pons and cervico-dorsal spinal cord, using [125I]PACAP-27 (PACAP 1-27). The tracer bound rapidly, specifically and reversibly. Competition binding curves were compatible with the coexistence, in the eight central nervous areas explored, of high and low affinity binding sites for PACAP-27 (Kd of 0.2 nM and 3.0 nM, respectively), and of only one class of binding sites for PACAP-38 (PACAP (1-38), Kd 0.2-0.9 nM). VIP inhibited only partially the binding of [125I]PACAP-27, and PHI, GRF(1-29)NH2 and secretin were ineffective at 1 microM. Chemical [125I]PACAP-27 cross-linking revealed a single specific 64 kDa protein species. In rat brain cortical membranes, saturation and competition experiments, using [125I]PACAP-38 as radioligand, indicated the presence of both high (Kd 0.13 nM) and low (Kd 8-10 nM) affinity binding sites for PACAP-38 and of low affinity (Kd 30 nM) binding sites for PACAP-27. These data taken collectively suggest the coexistence of PACAP-A receptors with a slight preference for PACAP-27 over PACAP-38 and of PACAP-B receptors that recognize PACAP-38 with a high affinity and PACAP-27 with low affinity. Both PACAP-27 and PACAP-38 stimulated adenylate cyclase with similar potency and efficacy. VIP was markedly less potent in this respect and also less efficient, except on cerebellar membranes.

Exocrine pancreas: evidence for topographic partition of secretory function

The pattern of amylase, lipase, and chymotrpsinogen content found in pancreatic exocrine tissue surrounding the islets of Langerhans (periinsular halos) differs from that of the rest, or teleinsular part, of the pancreas. Such a topographic partition of secretory function may play a role in the regulation of pancreatic juice composition.

The C-terminus ends of secretin and VIP interact with the N-terminal domains of their receptors

C-terminally truncated secretin and VIP molecules were synthesized, and their ability to occupy the recombinant secretin and VIP1 receptors stably expressed in Chinese hamster ovary (CHO) cells and to stimulate adenylate cyclase activity was studied. On secretin receptors, secretin (1-26) and secretin (1-24) were 10- and 50-fold less potent but as efficient as secretin (1-27); VIP (1-27) was as potent and efficient as VIP (1-28), and VIP (1-26) and VIP (1-25) were both 100-fold less potent. On VIP1 receptor, VIP (1-28) and VIP (1-27) were equipotent and VIP (1-26) and VIP (1-25) were 10- and 300-fold less potent, respectively; secretin (1-27) and secretin (1-26) were of equally low affinity and 10-fold more potent than secretin (1-24). Thus, the secretin and the VIP1 receptors had different selectivity profiles for the recognition of C-terminally truncated secretin and VIP derivatives. The chimeric receptors consisting in the N-terminal part of the secretin receptor on the core of the VIP1 receptor (N-Sn/VIP1.r) and in the N-terminal part of the VIP1 receptor on the core of the secretin receptor (N-VIP1/Sn.r) exhibited the selectivity pattern of the secretin and VIP1 receptors, respectively. The results suggest that the C-terminal end of secretin and VIP interacts with the N-terminal domain of the secretin and VIP receptors.

Fragments of pituitary adenylate cyclase activating polypeptide discriminate between type I and II recombinant receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) analogues were tested for their ability to occupy the recombinant selective PACAP receptors (PACAP type I receptor) or the non-selective PACAP-vasoactive intestinal polypeptide (VIP) receptors (PACAP type II, VIP1 and VIP2 receptors) stably transfected and expressed in Chinese hamster ovary (CHO) cells. The synthetic analogues consisted of N- and/or C-terminally shortened peptides. Thus, peptides starting at amino acid 1, 2, 3 or 6 and terminating at amino acid 27, 29, 30, 32 or 38 were compared on the three receptors studied. The shortening of PACAP-(1-38) to PACAP-(1-27) was of little influence. However, in N-terminally deleted peptides the PACAP-38 derivatives were of higher affinity than the PACAP-27 fragments on PACAP type I and PACAP type II, VIP2 receptors but not on PACAP type II, VIP1 receptors. The presence of the sequence 28-32 was in all cases sufficient to reproduce the data obtained with the PACAP-38 analogues. PACAP-(3-32) is able to discriminate the PACAP type II, VIP2 subtype from the other two subtypes, and PACAP-(6-30), PACAP-(6-32) and PACAP-(6-38) can discriminate the PACAP type II, VIP1 receptors from the other two subtypes. These molecules may help in the quantitative detection of receptor subclasses in complex systems when two or more receptor subtypes are found.

Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP- and secretin-like properties (helodermin) from Gila monster venom

A combination of three HPLC procedures applied to the venom of Gila monster (Heloderma suspectum) has led to the purification to homogeneity of two bioactive components: (i) a 17.5 kDa protein, isolated on the basis of its potent secretory effect on dispersed rat pancreatic acini, was accordingly designated PSF (pancreatic secretory factor); (ii) a 5.9‐kDa peptide, designated helodermin, was purified on the basis of its ability to stimulate adenylate cyclase in rat pancreatic membranes. PSF was unable to activate adenylate cyclase and, conversely, helodermin was devoid of secretory action.

α-Amylase et lipase du pancreas de rat. Purification chromatographique, recherche du poids moleculaire et composition en acides amines

Abstract 1. 1.|A rapid and efficient method of preparation of the cationic enzymes of the rat exocrine pancreas is described. The hydrolases are solubilized quantitatively and afterwards chromatographed on 2 coupled columns of Sephadex G-25 and DEAE-cellulose in 13 mM Tris-HCl buffer (pH 8.2). Filtration on Sephadex G-100 separates 7 or 8 constituents and among them are amylase and lipase. The latter enzymes are purified on Bio-Gel P-60 and on CM-cellulose respectively, after which they show homogeneity by disc electrophoresis. 2. 2.|Amylase and lipase constitute approx. 10.9% and 0.4% of the pancreatic proteins in rats fed a balanced diet. Starting from 30 animals (15 g of tissue) a yield of about 100 mg of amylase and 4 mg of lipase is usually obtained within 2 days after a 10-fold purification of the first enzyme and a 140-fold purification of the second. 3. 3.|Amylase has an excess of 3.5 basic residues % over acidic residues, contains 3.1% of tryptophan and has a E1 cm1 % value of 20 at 280 mμ. The specific activity is 600 units/mg at 25°. Amylase is increasingly adsorbed on Sephadex and Bio-Gels, when the porosity of these resins increases and when the ionic strength of the buffer decreases. Lipase has an excess of 3.2 basic residues % over acidic residues, a E1 cm1 % value of 12 at 280 mμ and a specific activity of 2500 units/mg at 25°. The molecular weight, determined by filtration on Sephadex G-100 and Bio-Gel P-150, is approx. 43 000. This value includes 14% of lipids made of free fatty acids (37%), phospholipids (35%), cholesterol esters (20%) and glycerides (8%). Lauric, myristic and palmitic acids are prominent in these lipids. 4. 4.|The rapid and efficient recovery of rat pancreatic amylase indicates the usefulness of a method that would be valid for other amylases: after filtration of a crude extract on a Sephadex-retaining amylase, this enzyme is filtered on a Bio-Gel from which it is more rapidly excluded than in the preceding filtration. Pig pancreatic amylase has been easily purified in this manner with an efficiency of 80%.

The two forms of the pituitary adenylate cyclase activating polypeptide (PACAP (1–27) and PACAP (1–38)) interact with distinct receptors on rat pancreatic AR 4‐2J cell membranes

The existence of specific receptors for the two PACAPs (Pituirary Adenylate Cyclase Activating Peptides of 27 and 38 amino acids) was previously demonstrated on membranes from the pancreatic acinar cell line AR 4‐2J (Buscail et al., FEBS Lett. 202, 77–81, 1990) by [125I]PACAP‐27 binding. Here we demonstrate, by comparing Scatchard analysis of saturation curves and competition binding curves obtained with [125I]PACAP‐27 and [125I]PACAP‐38 as radioligands, the coexistence of two classes of receptors : 1/ PACAP‐A receptors that recognize PACAP‐27 and PACAP‐38 with the same high affinity (K d 0.3 nM) and 2/ PACAP‐B receptors that recognize PACAP‐38 with a high affinity (K d 0.3 nM) and PACAP‐27 with a lower affinity (K d 30 nM). These two receptors are coupled to adenylate cyclase but can be clearly distinguished by the ability of PACAP(6–27) to specifically inhibit PACAP‐27 adenylate cyclase activation.