Jean Rathe

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.

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.

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.

Interaction of lipophilic VIP derivatives with recombinant VIP1/PACAP and VIP2/PACAP receptors

Stearyl vasoactive intestinal polypeptide has been reported to be a VIP (vasoactive intestinal polypeptide) receptor agonist of high potency with an original bioavailability and action. We synthesized three fatty acyl derivatives, myristyl-, palmityl- and stearyl-[Nle17]VIP, and tested their capacity to recognize recombinant rat- and human VIP1- and VIP2/PACAP (pituitary adenylate cyclase-activating polypeptide) receptors and to stimulate adenylate cyclase activity. The three lipophilic analogues bound with high affinity (from 0.5 to 20 nM) to both receptor subtypes but did not distinguish between them. In preparations expressing a high density of human VIP1/PACAP receptors, the three lipophilic analogues had the same efficacy as VIP and [Nle17]VIP. In preparations expressing the rat receptors, stearyl-[Nle17]VIP had a lower efficacy than the other peptides tested. In preparations expressing a low level of VIP1/PACAP receptors and in those expressing VIP2/PACAP receptors, all analogues behaved like partial agonists. The lowest efficacy was observed for stearyl-[Nle17]VIP on the VIP2/PACAP receptor subclass. Based on our results, a complex pattern of in vivo biological effects of the lipophilic VIP derivatives should be expected: these compounds might behave as full agonists, partial agonists, or antagonists of the VIP response, depending on the number and the subtype of receptor expressed.

Vasoactive intestinal peptide modification at position 22 allows discrimination between receptor subtypes

Secretin and growth hormone releasing factor (GRF) have a weak affinity for VIP (vasoactive intestinal peptide)/PACAP (pituitary adenylate cyclase activating polypeptide) receptors, but discriminate between VIP1/PACAP and VIP2/PACAP receptors. This previously allowed us to develop modified secretin and GRF derivatives as high affinity and highly selective VIP1/PACAP receptor ligands. We tested the hypothesis that the presence of a Gln residue at position 24 and a Leu residue at position 22 was responsible for their VIP1/PACAP receptor selectivity. [Gln24]VIP was not different from VIP but [Leu22]VIP had a 100-fold lower affinity for VIP2/PACAP receptors as compared to VIP1/PACAP receptors. The substitution of Tyr22 by Phe22 in VIP had no significant effect on the recognition of both receptors but [Ala22]VIP had a reduced affinity for the VIP2/PACAP receptor. This indicated that an aromatic residue at position 22 of VIP was required for a high affinity for the VIP2/PACAP receptor but not for the VIP1/PACAP receptor.

Isolation and primary structure of rat secretin

A major form of rat secretin was purified to homogeneity from small intestine, being detected with a porcine secretin radioimmunoassay throughout 7 chromatographic steps. The sequence of the heptacosapeptide amide H-S-D-G-T-F-T-S-E-L-S-R-L-Q-D-S-A-R-L-Q-R-L-L-Q-G-L-V-NH2 shows that rat secretin has a glutamine residue in position 14 instead of arginine as in pig secretin.

C-Terminally shortened pituitary adenylate cyclase-activating peptides (PACAP) discriminate PACAP I, PACAP II-VIP1 and PACAP II-VIP2 recombinant receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) analogues were tested for their ability to occupy the recombinant selective PACAP receptors (PACAP type I receptors) and the non-selective PACAP-vasoactive intestinal polypeptide (VIP) receptors (PACAP type II, VIP1 and PACAP type II, VIP2 receptors), stably transfected and expressed in Chinese hamster ovary cells. Their capacity to stimulate the adenylate cyclase activity was also measured. The synthetic analogues tested were peptides shortened at the carboxyl terminus by the removal of 1-4 amino acids (PACAP-26 to PACAP-23). All the peptides discriminated the 3 receptor subtypes and had the highest affinity for the VIP1 receptors, and the lowest affinity for the VIP2 receptors; PACAP-25 having the highest ability to discriminate the VIP1 and VIP2 receptors. All the peptides tested were full agonists on the PACAP I and VIP1 receptors; PACAP-25 and -26 were partial agonists on VIP2 receptors and may be appropriate tools to establish the receptor subtype involved in a given cellular response.

Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using 125I-CDR as a tracer molecule. 125I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca2+ and Mg2+ dependence was observed for 125I-CDR binding and for brain adenylate cyclase activation by CDR.