Denis Gossen

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.

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.

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.

Characterization and regulation of atrial natriuretic peptide (ANP)-R1 receptors in the human neuroblastoma cell line NB-OK-1

We characterized in membranes from the human neuroblastoma cell line NB-OK-1, an ANP-R1 receptor (Mr 130 kDa) for the atrial natriuretic peptide (ANP). This receptor recognized biologically active forms of ANP with high affinity but showed no affinity for truncated ANP forms. It was functional in that binding correlated with guanylate cyclase activation (a 2-fold increase in Vmax) with the following rank order of potency: rat ANP-(99-126) greater than human ANP-(99-126) greater than human ANP-(102-126) greater than porcine BNP (brain natriuretic peptide). The enzyme required free Mn2+ in addition to the Mn-GTP substrate (Km of about 0.3 mM for both basal and ANP-stimulated activity). In the presence of dithiothreitol, the dose-response curve of guanylate cyclase activation was shifted rightward by a factor of 30. ANP-R1 receptors were upregulated through protein synthesis in cells exposed to 1 mM carbamylcholine or 1 mM dibutyryl cyclic AMP for 8-24 h (ANP was ineffective).

Molecular architecture of secretin receptors: The specific covalent labelling of a 51 kDa peptide after cross-linking of [125I]iodo-secretin to intact rat pancreatic acini

p‐Azidophenylglyoxal (APG), a heterobifunctional reagent with one group reacting selectively with arginine residues and another group photoactivable, was used to cross‐link [125I]secretin prebound to intact rat pancreatic acini. The best yield was obtained when the [125I]secretin‐acini complex was incubated under dim light with 2 mM APG at 37°C and pH 8.0, followed by photolysis at 312 nm. The main secretin binding peptide cross‐linked under reducing conditions, when tested by SDS‐PAGE and autoradiography: (i) had a molecular mass of 51 kDa and was not a subunit of a larger disulfide‐linked structure, and (ii) was distinct from the main VIP binding peptide coexisting in the same preparation.

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1

125I‐[Tyr2]scyllatoxin allowed to label a single class of high‐affinity receptors in membranes from the human neuroblastoma cell line NB‐OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the B max was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5‐fold after a 24‐h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.

Assays for Secretin Receptors: Comparison between Neuroblastoma Cells and Exocrine Pancreas

Publisher Summary This chapter focuses on the investigative methods that use assays to characterize secretin receptors. The secretin mRNA in the gut of rat and pig is deduced, and it is found to be present in rat tissues in the following decreasing order of concentration: ileum>duodenum>colon. Pancreatic plasma membranes are prepared from Wistar albino rats, and 2-mercaptoethanol is omitted from all buffers owing to the lability of secretin receptors in the presence of this reducing agent. Synthetic porcine secretin is radioiodinated at the level of the N-terminal histidine residue, at basic pH, by the chloramine-T method. The receptor to total protein ratio being small, the cross-linking yield is optimized to obtain significant autoradiographic signals without overloading sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. SDS-PAGE is performed using a discontinuous system buffer with an electrophoresis buffer concentration twice that mentioned in the original method. Gel calibration is performed with standard protein kits from Pharmacia—Piscataway, NJ—and Bio-Rad—Richmond, CA. The conditions used probably maintain intact the plasma membranes of dispersed acini. However, the yield is very low, and more than one month exposure is necessary to visualize the cross-linked secretin.