Effect of introduction of an arginine16 in VIP, PACAP and secretin on ligand affinity for the receptors.

Rabbit secretin, which differs from all other mammalian secretins in having a Leu residue in position 6 (instead of Phe) and a basic residue (Arg) in position 16, had a lower affinity than porcine secretion on recombinant rat secretin receptors but had a greater affinity than porcine secretin on recombinant rat VIP1 and PACAP I receptors. Synthetic [L6] porcine secretin had a reduced potency on secretin and VIP1 receptors whereas [R16] porcine secretin had a similar binding profile as rabbit secretin. Thus, an arginine residue in position 16 reduced 3-fold the affinity of secretin for secretin receptors but increased 30-fold its affinity for the VIP1 and PACAP I receptors. The introduction of an arginine residue in position 16, instead of glutamine, in VIP and PACAP had a similar effect: [R16] VIP and [R16] PACAP had 3- to 10-fold higher affinities than VIP and PACAP for VIP1 and PACAP I receptors, and 3-fold lower affinities for the secretin receptors. The three [R16] peptides also had a reduced potency on the chimeric receptor consisting of the N-terminal part of the secretin receptor grafted on the VIP1 receptor, and an enhanced potency on the chimeric receptor consisting of the N-terminal part of VIP1 receptor grafted on the secretin receptor, indicating that position 16 of each ligand interacted with the N-terminal extracellular domain of the receptors.

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide (1-27) and secretin from the small intestine of guinea pig.

The neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucinamide (1-27) (PHI) and the hormone secretin were purified from the small intestine of guinea pig, being detected by radioimmunoassay and radioreceptor assay throughout six to seven chromatographic steps. After elution on a reverse-phase C18 column, the three peptides were separated on a Fractogel column. After cation-exchange chromatography of each peptide on Mono S, the final steps were performed using a reverse-phase RP8-e column. Guinea pig PHI differed from porcine PHI in having Tyr and Arg residues instead of Phe and Lys in, respectively, position 10 and 20. We confirmed the original sequence of guinea pig VIP previously documented (with Leu5, Thr9, Met19 and Val26). We also established the similarity of the primary structure of guinea pig secretin with that of porcine and bovine.

The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes.

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts.

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J.

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).

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 (Pituitary 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 (Kd 0.3 nM) and 2/PACAP-B receptors that recognize PACAP-38 with a high affinity (Kd 0.3 nM) and PACAP-27 with a lower affinity (Kd 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.

The sensitivity of dot immunoassay for the peptides helodermin, histidine-isoleucinamide (PHI) and histidine-methioninamide (PHM) increases after peptide cross-linking to proteins prefixed on nitrocellulose.

Two out of three rabbit anti-helodermin antisera previously shown to be useful for radioimmunoassay failed to detect up 100 ng of the peptide helodermin spotted directly on a nitrocellulose membrane. A lesser shortcoming of dot immunoassay was encountered with porcine PHI (peptide histidine-isoleucinamide), a member of the same peptide family, and an anti-PHI antiserum. To improve antigen-antibody interactions in the solid phase, we compared five methods of prior peptide immobilization. The best result was obtained when the nitrocellulose membrane was pretreated for 1 min in 4% ovalbumin, followed by 10 min activation with 2.5% glutaraldehyde, before peptide spotting. After cross-linking, the peptide was immunodetected with a F(ab')2 fragment of an anti-rabbit IgG coupled to alkaline phosphatase. The peptide cross-linking method was capable of increasing the sensitivity of ensuing immunodetection by more than 1000-fold, i.e., made feasible the detection of 0.1 ng peptide/dot in cases when the direct spotting method was inefficient. The sensitivity of this new, reliable and simple dot immunoassay for peptides was comparable to the conventional dot assay of directly immobilized large proteins.

Structural requirements for the occupancy of rat brain PACAP receptors and adenylate cyclase activation.

N-terminally shortened analogues of PACAP(1-27) and PACAP(1-38), and analogues modified in position 1,2 or 3 were compared for their ability to interact with PACAP receptors and to activate or inhibit adenylate cyclase in rat brain hippocampus membranes. In the PACAP(1-27) series, deletion of the first two amino acids decreased the potency 3000-fold. PACAP fragments (3-27) to (9-27) were inactive on the enzyme. N-terminally shortened PACAP(1-38) analogues showed a similar profile but were 70 to 300-fold more potent than their PACAP(1-27) equivalents. PACAP(6-27) and PACAP(6-38) were competitive inhibitors of the PACAP(1-27) stimulated enzyme. The Kd values of PACAP(6-27) and PACAP(6-38) were of 1000 and 2 nM respectively. Surprisingly, the Kd values of PACAP(6-31) and (6-35), that were also unable to stimulate adenylate cyclase activity, were of 3 and 300 nM respectively. Replacement of His1 by Phe1 in PACAP(1-27) reduced the potency 600-fold. Replacement of Ser2 by Ala2 in PACAP(1-27) and PACAP(1-38) was of little consequence. Substitution of Ser2 by Phe2, DPhe2, Arg2 or DArg2 reduced 60 to 1000-fold the PACAP(1-27) potency but only 7 to 30-fold the PACAP(1-38) potency. Phe2 derivatives were inactive on the enzyme. Replacement of Asp3 by Asn reduced 4000-fold the PACAP(1-27) potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP-27, but not PACAP-38) degradation by the neutral endopeptidase EC 3.4.24.11.

VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase-activating polypeptide), which are potent relaxing agents in the airways, were submitted to in vitro degradation by the neutral endopeptidase EC 3.4.24.11 (NEP), one of the most active peptidase in the lung, to test their relative resistance to proteolysis. Both VIP and PACAP(1-27) were cleaved by NEP, but PACAP(1-38) was not. The main fragments produced were VIP(1-22) and VIP(1-25), and PACAP(1-22) and PACAP(1-25), respectively. The degradation of VIP(1-27), PACAP(6-27), and PACAP(13-27) was also hindered by extending their C-terminal ends with the (28-38) sequence of PACAP(1-38). The sensitivity to enzyme degradation was gradually reduced when the C-terminal extension was increased from PACAP(1-27) to PACAP(1-29), PACAP(1-32) and PACAP(1-38). The biological activities of the degradation products were evaluated on the three classes of PACAP/VIP receptors, with VIP(1-25) and PACAP(1-25) retaining an important part of their activities on the VIP1 receptor. Thus, the degradation of VIP and PACAP(1-27) by the neutral endopeptidase 24.11 might produce a VIP1 receptor-selective active metabolite, provided that very high VIP or PACAP(1-27) concentrations are achieved in the receptor vicinity.

Inhibition of Na/Ca exchange by Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides in intact rat pancreatic B-cells.

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides were recently shown to inhibit Na/Ca exchange in cardiac sarcolemmal vesicles. In the present study, we examined the effects of FMRFa-related peptides on Na/Ca exchange in intact (pancreatic B) cells. At 2.8 mM glucose, FMRFa-related peptides only weakly inhibited Na/Ca exchange although their effect was more marked under depolarizing conditions. The peptides blocked neither the Na/K-ATPase nor Ca2+ channels but slightly reduced membrane K+ permeability. Our data indicate that FMRFa-related peptides are weak and non-specific inhibitors of Na/Ca exchange in intact B cells. The data do not confirm the view that the peptides may exert some of their physiological modulatory role by inhibiting Na/Ca exchange.

Analogues of VIP, helodermin, and PACAP discriminate between rat and human VIP1 and VIP2 receptors.

Vasoactive intestinal polypeptide (VIP) acts through interaction with two subclasses of seven transmembrane G protein-coupled receptors named VIP1 and VIP2 receptors. These receptors have been cloned in different species, such as rat and human. Considering the different distribution of both receptor subclasses, there is considerable interest in the development of selective agonists and antagonists. The present study compares the binding properties of VIP, PACAP, GRF, secretin, and helodermin analogues on recombinant rat and human VIP1 and VIP2 receptors. On both rat and human receptors, secretin and GRF had a higher affinity for the VIP1 receptor subtypes. The amino-shortened VIP, and the carboxy terminal-shortened VIP and PACAP analogues also presented a higher affinity for the VIP1 receptor. PHI, PHV, helodermin, and helospectin were selective for the human VIP2 receptor subtypes. These results suggest that the helical structure of the carboxy terminal end is necessary for VIP2 recognition. The differences between species were the following: PHI, PHV, helodermin, and helospectin had a higher affinity for the rat VIP1 receptor than for the human VIP1 receptor. On both rat and human receptors, D-Ala4 VIP and D-Phe4 VIP had a high affinity for the VIP1 receptor and a low affinity for the VIP2 receptor. Thus, three domains of the ligand involved in VIP1/VIP2 receptor discrimination were identified: the amino acid residue in position 4 ([D-Ala4], [D-Phe4]VIP), in positions 8 and 9 (the effects of helodermin and helospectin), and the carboxy terminal end (the effects of the shortened VIP and pituitary adenylate cyclase activating polypeptide analogues).

Autoradiographic visualization of the receptor subclasses for vasoactive intestinal polypeptide (VIP) in rat brain.

Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP1- and the VIP2 receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretion analogue ([R16]chicken secretin) were identified as selective agonist peptides for the VIP2- and VIP1 receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP1 receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP2 receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon's horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.

In vitro properties of a high affinity selective antagonist of the VIP1 receptor.

A selective high affinity VIP1 receptor antagonist [Acetyl-His1, D-Phe2, Lys15, Arg16, Leu17] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP1 receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP1-, rat VIP2- or the human VIP2-recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP1 receptor. The IC50 values of 125I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP1-, rat VIP2-, human VIP1- and human VIP2 receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP1 receptor mediated stimulation of adenylate cyclase activity. The Ki values were respectively of 15 +/- 5 nM and 2 +/- 1 nM for the rat and human VIP1 receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP1 receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.

Comparative in vitro effects of guinea pig VIP and common VIP on liver and lung membranes from guinea pig and rat and on human lymphoblastic SUP-T1 membranes.

Guinea pig VIP differs from VIP of several mammals by its amino acids in positions 5, 9, 19 and 26. We tested a) its ability to occupy VIP receptors in liver and lung membranes of rat and guinea pig and in the human lymphoblastic SUP-T1 cell line and b) the ensuing adenylate cyclase stimulation. In liver and lung membranes from rat, guinea pig VIP was less potent than common VIP to occupy high and low affinity VIP receptors. In rat liver both VIP activated adenylate cyclase mostly through high affinity receptors. In rat lung, guinea pig VIP activated the enzyme mostly through high affinity receptors and was less efficient than common VIP acting through both classes of receptors. In guinea pig liver and lung membranes, binding inhibition curves were steeper than with rat preparations and adenylate cyclase appeared to be mostly activated through high affinity VIP receptors in liver and through both classes of receptors in lung. On human lymphoblastic SUP-T1 membranes both VIP were equally potent and efficient to inhibit tracer binding and activate adenylate cyclase.

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.

Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC(2) receptor.

Ro 25-1553 is a cyclic VIP derivative with a high affinity for the VPAC(2) receptor subtype. Our goal was to identify the modifications that support its selectivity for VPAC(2) receptors, and to develop a VIP or Ro 25-1553 analog behaving as a high affinity, VPAC(2) selective antagonist. The selectivity of Ro 25-1553 for the human receptor was supported mainly by the acetylation of the amino-terminus, by the introduction of a lysine residue in position 12, and by the carboxyl-terminal extension. The lactam bridge created between positions 21 and 25 contributed to the affinity of the compound for the VIP receptors but participated only marginally to its selectivity. Deletion of the first five aminoacid residues led to a low affinity antagonist with a low selectivity. Introduction of a D-Phe residue in position 2 reduced the affinity, the selectivity and the intrinsic activity, the compound being a partial agonist. Myristoylation of the amino-terminus of [K(12)]VIP(1-26) extended carboxyl-terminally with the -K-K-G-G-T sequence of Ro 25-1553 led to a high affinity, selective VPAC(2) receptor antagonist. This molecule represents the first selective human VPAC(2) receptor antagonist described to date.

Evidence that the chromogranin B fragment 368-417 extracted from a pheochromocytoma is phosphorylated.

A rabbit antiserum was raised against a synthetic peptide corresponding to residues 403 to 417 of human chromogranin B. This peptide was chosen to match the potential C-terminal end of a putative proteolytic fragment of the protein located between dibasic doublets in positions 366-367 and in positions 418-419 of the precursor. A radioimmunoassay based on this antiserum was developed and used to detect the protein or a fragment thereof in a pheochromocytoma tumor extract. One fragment was purified to homogeneity by successive reverse-phase HPLC chromatographies. The N-terminal sequence established by automated Edman degradation, was N-Y-P-S-L-E-L-D-K-M-A-H-G-Y-G-E-E-S-E-E-E-R corresponding to the 368-389 sequence of human chromogranin B. Taking into account the specificity of the antiserum used for peptide identification and alignment with the precursor sequence, we deduced that the purified peptide was chromogranin B (368-417) and represented a new peptide generated by limited proteolysis of chromogranin B. Combining electrospray mass-spectrometry and enzymatic dephosphorylation, we demonstrated that this peptide was phosphorylated.

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.

The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK.

We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide [PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version] to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. [125I]PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited [125I]PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.