Marie-Claire Vandermeers-Piret

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.

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.

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.

Effect of colipase on adsorption and activity of rat pancreatic lipase on emulsified tributyrin in the presence of bile salt

The present work was undertaken to examine rat pancreatic lipase activity in relation to its ability to be adsorbed on emulsified tributyrin. This study was conducted at a supramicellar concentration of sodium taurodeoxycholate, between pH 6.0 and 8.0, and at different colipase concentrations. A pH adsorption curve was differentiated from the pH activity curve of lipase. The authors concluded that the socalled acid shift of the optimal pH for lipase action described earlier is due to the low adsorption rate of lipase on its substrate at alkaline pH rather than to a change of the pH dependence of the V(max) and K(m) of the enzyme.

On human pancreatic triacylglycerol lipase: Isolation and some properties

Abstract A single triacylglycerol lipase (EC 3.1.1.3) containing approx. 420 amino acid residues has been purified from human pancreatic juice with a 30% overall yield. The method involved column chromatography on Sephadex G-25, diethylaminoethyl-cellulose, carboxymethyl-cellulose and Sephadex G-75. The molecular weight of the enzyme was estimated to be 46 000 from the amino acid composition. The purified preparation was incapable of catalyzing a prolonged hydrolysis of tributyrin at 25 °C, because of rapid inactivation. The addition of sodium taurodeoxycholate exerted a protective effect on lipase, an inhibitory action on lipolysis, and a shift of the optimal pH down to pH 7.5. Increasing the NaCl concentration enhanced the inhibitory effect of the bile salt, whereas bovine colipase and serum albumin prevented this effect. Only colipase was able to ensure a maximal reaction rate, both in the presence or absence of sodium taurodeoxycholate. Our data indicate that each molecule of human triacylglycerol lipase can easily react with one molecule of bovine colipase, and less easily with two or more molecules.

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as effectively as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.

Immunoreactive helodermin-like peptides in rat: a new class of mammalian neuropeptides related to secretin and VIP.

Helodermin is a peptide from the venom of the lizard Heloderma suspectum (Gila Monster) showing a high degree of sequence similarity with VIP, PHI and secretin in its N-terminal moiety. The present data support the presence of peptide(s) closely related to helodermin in the brain, gut and salivary glands of rat. In our radioimmunoassays, we routinely used one of the three specific antisera obtained from rabbits that were immunized against lizard helodermin coupled to bovine serum albumin with carbodiimide. Heat- and acid-stable immunoreactive helodermin-like material was more abundant in striatum, hippocampus and anterior pituitary than in cerebral cortex and hypothalamus. High levels of helodermin-like material were also present in salivary glands, duodenum and jejunum. When submitted to gel permeation chromatography on a TSK-G 2000 SW column, the apparent molecular radius of most of the immunoreactive material ranged from 6 to 12 KDa.