Annick Cauvin

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 (K d =0.4 nM) and PACAP‐38, while their affiity for VIP and the parent peptide helodemin was 1000‐fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP‐38 and PACAP‐27 (K act = 0.2 nM) being much higher than that of VIP (K act= 100 nM) and helodemin (K act = 30 nM). Chemical cross‐linking of [125I]PACAP‐27 followed by SDS‐PAGE and autoradiography revealed a specifically cross‐linked peptide with an M r, of 68000 (including 3000 for one PACAP‐27 molecule).

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.

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.

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.

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.

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.

Amino acid sequence of VIP, PHI and secretin from the rabbit small intestine

VIP, PHI and secretin were purified from rabbit small intestine throughout a maximum of 6 chromatographic steps. After elution on a reverse phase C18 column, the 3 peptides were separated on a Fractogel column using specific radioimmunoassays for detection. After cation exchange chromatography on Mono S, the final steps were performed using a reverse phase RP8-e column. For these steps, radioreceptor assays were utilized to detect VIP and PHI. We confirmed that the VIP sequence of rabbit was identical to that of porcine VIP. The PHI sequence was also found identical to that of porcine PHI. By contrast, rabbit secretin was highly original, differing from porcine secretin in having Leu, Arg and Leu-NH2 residues instead of Phe, Ser and Val-NH2 in, respectively, position 6, 16 and 27.

Rat PHI, PHI-GLY and PHV(1-42) stimulate adenylate cyclase in six rat tissue and cell membranes

PHI and the two C-terminally extended forms PHI-GLY and PHV(1-42) coexist in rat tissues. We compared the relative potency and efficacy of these three PHI forms and of VIP to stimulate adenylate cyclase activity and, when feasible, to occupy VIP receptors in six rat tissue and cell membranes. With the exception of lung membranes, all three PHI forms were markedly less potent than VIP but all were systematically as efficacious. PHI-GLY and PHV(1-42) were never more potent than PHI itself and their relative potencies revealed four spectra, depending on the membrane preparation tested: 1) PHI = PHI-GLY = PHV(1-42) in hepatic, pulmonary and pancreatic membranes; 2) PHI greater than PHV(1-42) = PHI-GLY in membranes from circulating lymphocytes; 3) PHI = PHV(1-42) greater than PHI-GLY in membranes from the thymocyte cell line 51E; and 4) PHI greater than PHI-GLY = PHV(1-42) in anterior pituitary membranes. These results indicate that the two naturally observed C-terminal extensions of rat PHI variously affected peptide potency on 6 rat membrane preparations.

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.

VIP Receptors in Human SUP-T1 Lymphoblasts

We characterized a new type of vasoactive intestinal peptide (VIP) receptors in the CD4+ Stanford University Pediatric (SUP)-T1 lymphoma cell line, by comparing receptor occupancy [in the presence of (125I)helodermin and (125I)(acetyl-His1)VIP] and adenylate cyclase activation (in the presence of GTP). The order of potency of peptides on both parameters was: helodermin greater than (acetyl-His1)VIP greater than (Phe1)VIP = VIP greater than PHI while secretin was ineffective. In membranes, when Gs was permanently activated by Gpp(NH)p or by ADP-ribosylation (after pretreating intact lymphoblasts for 2 h with cholera toxin), there resulted a variably increased affinity of receptors for VIP-like peptides, suggesting reduced receptor selectivity. Preexposing intact lymphoblasts to the same peptides induced, within 5 min, homologous desensitization (i.e. reduced binding capacity and even more so impaired capability to activate adenylate cyclase), whose extent correlated with the Kd of each peptide at time 0. After prolonged (16 h) exposure to 30 nM VIP that resulted in marked (75%) downregulation, 60% of the adenylate cyclase responsiveness could recover within 30-120 min even in the presence of cycloheximide, but further resensitization was cycloheximide-sensitive. To conclude, VIP receptors coupled to adenylate cyclase showed distinct specificity in human SUP-T1 lymphoblasts. Their specificity decreased when Gs was permanently activated. In intact cells exposed to VIP-like peptides, the receptors were rapidly desensitized, then down-regulated, the resensitization mechanism being not immediately inhibited by cycloheximide.