Comparison of biologic porcine secretin, synthetic porcine secretin, and synthetic human secretin in pancreatic function testing.

BACKGROUND AND AIMS: Due to the unavailability of biologic porcine secretin (BPS), 2 synthetic forms of secretin were developed. Our aim is to determine the bioequivalency of the 3 forms of secretin in pancreatic function testing. METHODS: In a randomized, crossover design, synthetic porcine (SPS) and synthetic human secretin (SHS) were compared in a group of 12 subjects with chronic pancreatitis undergoing secretin stimulation test (SST). The 2 synthetic forms of secretin were then compared with BPS in 12 subjects utilizing a similar design. Finally, 18 healthy subjects underwent secretin stimulation testing with SHS. RESULTS: There was excellent correlation of peak bicarbonate measurements in the comparison of SPS to SHS (R = 0.967) as well as in the comparison of all 3 forms of secretin (P = 0.08, ANOVA for correlated samples). In the SST, each of the synthetic forms of secretin were 100% accurate in diagnosing chronic pancreatitis in disease subjects and in excluding chronic pancreatitis in normal controls. The synthetic forms of secretin were associated with fewer side effects when compared with BPS with the exception of transient tachycardia which occurred in up to 19% of subjects. CONCLUSIONS: The synthetic porcine and human forms of secretin are equivalent to one another and to biologic porcine secretin and can be used interchangeably in pancreatic function testing.

Development of a biologically active secretin analogue incorporating a radioiodinatable photolabile p-(4-hydroxybenzoyl)phenylalanine in position 10.

Photoaffinity labeling is a powerful approach for direct elucidation of residue-residue approximations as a ligand is bound to its receptor, providing important constraints for molecular modeling. Probes utilized for this need to incorporate photolabile sites of covalent attachment and an indicator, such as a radiolabel. Radioiodine provides a particularly useful high specific radioactivity label, but due to its size, can only be accommodated in limited positions within a peptide ligand. In this work, we attempted to develop a probe for the secretin receptor that would directly provide spatial approximation data for position 10 of secretin, its site of radiolabeling. This was achieved by incorporation into a secretin analogue of the radioiodinatable and photolabile benzophenone moiety, p-(4-hydroxybenzoyl)phenylalanine (OH-Bpa). An unintended additional modification of secretin in synthesizing this probe was the elimination of Gly(4). This probe was shown to bind to the secretin receptor specifically and saturably (K(i)=25.3+/-6.0 nM). It represented a full agonist, stimulating intracellular cAMP in a concentration-dependent manner (EC(50)=4.2+/-0.7 nM). It was also able to affinity label the secretin receptor in a specific and efficient manner. This probe should provide the opportunity to identify the region of the secretin receptor in spatial approximation with position 10, within the pharmacophore of secretin, leading to refinement of molecular conformational models of this agonist-bound receptor.

Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes.

Only one secretin receptor has been cloned and its properties characterized in native and transfected cells. To test the hypothesis that stimulatory and inhibitory effects of secretin are mediated by different secretin receptor subtypes, pancreatic and gastric secretory responses to secretin and secretin-Gly were determined in rats. Pancreatic fluid secretion was increased equipotently by secretin and secretin-Gly, but secretin was markedly more potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with the rat secretin receptor, secretin and secretin-Gly equipotently displaced (125)I-labeled secretin (IC(50) values 5.3 +/- 0.5 and 6.4 +/- 0.6 nM, respectively). Secretin, but not secretin-Gly, caused release of somatostatin from rat gastric mucosal D cells. Thus the equipotent actions of secretin and secretin-Gly on pancreatic secretion appear to result from equal binding and activation of the pancreatic secretin receptor. Conversely, secretin more potently inhibited gastric acid secretion in vivo, and only secretin released somatostatin from D cells in vitro. These results support the existence of a secretin receptor subtype mediating inhibition of gastric acid secretion that is distinct from the previously characterized pancreatic secretin receptor.

COOH-terminally extended secretins are potent stimulants of pancreatic secretion.

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and alpha-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 +/- 9% (P > 0.05) and 176 +/- 13% (P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.

Synthesis of biologically active porcine secretin and [ITyr10] porcine secretin.

Porcine secretin, [Tyr10] secretin, and [Tyr13] secretin were synthesized by solid phase methodology and purified by stepwise gradient elution from a short reversed-phase column with ethanol and acetic acid as organic modifiers. [Tyr10] secretin and [Tyr13] secretin were iodinated by the chloramine-T method and nonmono-, and di-iodinated products separated and isolated by reversed-phase HPLC. Batch incubation analysis is isolated mouse pancreatic islets revealed that secretin and the [Tyr10] analogue were indistinguishable in their effect on the glucose-induced insulin release and cAMP accumulation. [Tyr13] secretin in contrast was significantly less potent in its effect on the glucose-induced insulin release.

Reduced peptide bond pseudopeptide analogues of secretin. A new class of secretin receptor antagonists.

The ability to assess the importance of secretin in various physiological processes is limited by the lack of specific potent antagonists. Recently, reduced peptide bond (psi) analogues of bombesin or substance P in which the -CONH- bond is replaced by -CH2NH- are reported to be receptor antagonists. To attempt to develop a new class of secretin receptor antagonists, we have adopted a similar strategy with secretin and sequentially altered the eight NH2-terminal peptide bonds, the biological active portion of secretin. In guinea pig pancreatic acini, secretin caused a 75-fold increase in cyclic AMP (cAMP). Secretin inhibited 125I-secretin binding with a half-maximal effect at 7 nM. Each of the psi analogues inhibited 125I-secretin binding. [psi 4,5]Secretin was the most potent, causing the half-maximal inhibition at 4 microM, and was 2-fold more potent than the [psi 1,2]secretin; 7-fold more than [psi 3,4]secretin, [psi 5,6]secretin, and [psi 8,9]secretin; 9-fold more than [psi 7,8]secretin; 13-fold more potent [psi 6,7]secretin, and 17-fold more than [psi 2,3]secretin. Secretin caused a half-maximal increase in cAMP at 1 nM. At concentrations up to 10 microM, [psi 2,3]secretin, [psi 4,5]secretin, and [psi 8,9]secretin did not alter cAMP whereas [psi 1,2]secretin and [psi 6,7]secretin caused a detectable increase in cAMP at 10 nM, [psi 7,8]secretin at 300 nM, [psi 5,6]secretin at 1 microM, and [psi 3,4]secretin at 10 microM. The [psi 4,5], [psi 2,3], and [psi 8,9] analogues of secretin each inhibited 1 nM secretin-stimulated cAMP as well as [psi 3,4]secretin, which functioned as a partial agonist. [psi 4,5]Secretin was the most potent, causing half-maximal inhibition at 3 microM whereas [psi 8,9]secretin was 6-fold less potent, and [psi 2,3]secretin and [psi 3,4]secretin were 17-fold less potent. [psi 4,5]Secretin inhibited secretin-stimulated cAMP and binding of 125I-secretin in a competitive manner. [psi 4,5]Secretin did not interact with cholecystokinin, bombesin, calcitonin gene-related peptide, or cholinergic receptors but did interact with receptors for vasoactive intestinal peptide, causing half-maximal inhibition at 72 microM and thus had a 18-fold higher affinity for secretin than vasoactive intestinal peptide receptors. These results indicate that reduced peptide bond analogues of the NH2 terminus of secretin represent a new class of secretin receptor antagonists. It is likely that in the future even more potent members of this class can be developed which may be useful to investigate the role of secretin in various physiological processes.

Side products in the synthesis of secretin.

Analytical studies on side products in the synthesis of secretin led to the discovery of a further side reaction, involving conversion of the carboxamide function of glutamine to glutamic acid gamma-methyl ester. The chemical structure of the side products was determined by NMR spectroscopy upon their tryptic digestion and isolation of the modified secretin fragments. Possible pathways for the observed side reaction are discussed.

Identification of secretin diastereoisomers produced during synthesis.

The byproducts P-1 and P-2, which were produced during the synthesis of porcine secretin, were isolated in pure form from the crude secretin by HPLC. These were identified by a combination of amino acid analysis, enzymatic digestion, and isocratic or linear gradient reversed-phase (RP)-HPLC. The amino acid compositions of P1 and P2, determined by amino acid analysis after acid hydrolysis, were found to be the same as those of porcine secretin without distinction between L-and D-amino acids. But, HPLC of their digestive fragments with trypsin and alpha-chymotrypsin differed from that of secretin. The fragments, S7-12 of P-1 and S13-21 of P-2 were determined to be different from the corresponding fragments obtained from secretin by HPLC analysis of their digestive fragments. The amino acid composition of each acid hydrolysate, following digestion with D-amino acid oxidase, was found to have less leucine or alanine content than secretin. The HPLC analysis of the fragments from P-1 and P-2 by tryptic and alpha-chymotryptic digestion showed that they are the same as those from synthetic D-Leu10 secretin or D-Ala17 secretin, respectively. Consequently, P-1 and P-2 are concluded to be the secretin diastereoisomers, D-Leu10 and D-Ala17 secretin, respectively.

Chemical synthesis and cloning of secretin gene.

A DNA duplex coding for the 27 amino acids of secretin has been synthesized and cloned. In designing the sequence of the gene, computer analysis has been applied. The following factors have been considered: selection of codon usage in favour of expression in yeast; design of various sites useful in gene cloning, gene modification and expressed product purification; avoiding the repeat sequences which may interfere in the ligation of the synthetic fragments. The synthesis involved preparation of 12 oligodeoxyribonucleotides (12-mer to 24-mer in length) by phosphate triester and phosphite triester method, purification by polyacrylamide gel electrophoresis (PAGE). A new plasmid pWS1 was constructed by insertion of the enzymatic ligated gene fragment into plasmid pWR13.

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs.

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.

New analogues of secretin.

For the evaluation of structure/activity relationships, some porcine secretin analogues, modified in the N-terminus, have been synthesized by segment condensation in solution. The secretin activity of the analogues was defined as the volume of pancreatic juice secreted in rats and dogs. The exchange of the N-terminal pentapeptide for the N-terminal pentapeptide of human somatotropin releasing factor (h-SRF) resulted in a peptide ([1-Tyr,2,4-Di-Ala,5-Ile]secretin) with practically no SRF-activity (less than 1% SRF-activity up to 100 micrograms/kg in the rat), but surprisingly high secretin activity (almost 100% in the rat, but only 1150 CU/mg (27%) in the dog). [3-L-Cysteic acid]secretin showed 1750 CU/mg (39%) in the dog, but a less activity (23%) in the rat. [6-D-Phe]secretin and [5-D-allo-Thr]secretin are again strongly species specific. They exhibited an activity of less than 1% in the dog, but about 10-15% in the rat. The smallest secretin activity was observed with [1-Cys,6-Cys]secretin in the oxidized form. The activity in the rat with this analogue was only about 0.2%.

Analogues and fragments of secretin.

For the purpose of analytical investigation and structure/activity relationships, some secretin analogues and secretin fragments have been synthesized. HPLC comparison of the synthesized products with our synthetic secretin revealed about 2% [D-Ala17]secretin, 1% [D-Leu13]secretin and less than 1% aminoterminal degradation products. The D-Ala17 content can be eliminated if the starting material used for segment coupling (Z-Arg(Z2)-Asp(OBut)-Ser(But)-Ala-OH) has no D-Ala-contamination. In addition, traces of the rearrangement products [3-aspartoyl]-secretin and [beta-Asp3]secretin are suspected. Secretin can be degraded to several compounds by chromatography on a strong basic ion exchanger in 1% acetic acid. These products are more polar than secretin and have no biological activity. The secretin content measured by HPLC correlated well with the biological data, since the degradation products and other byproducts separated by HPLC have only a negligible influence on the pancreatic flow.

Studies on peptides. CXIII. Synthesis of the heptacosapeptide amide corresponding to the entire amino acid sequence of chicken secretin.

The heptacosapeptide amide corresponding to the entire amino acid sequence of chicken secretin was synthesized by assembling four peptide fragments followed by deprotection with thioanisole-mediated trifluoromethanesulfonic acid in TFA. The deprotected peptide was purified by gel-filtration on Sephadex G-25, followed by ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose. Synthetic peptide stimulated the flow of pancreatic alkaline juice and decreased systemic blood pressure in rats.

Solid-phase synthesis and biological activities of gastrointestinal hormones: secretin and motilin.

Many successful solid-phase syntheses of peptide chains in the region of 20-40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparation high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as power lyophilized from dilute acetic acid.

Improvements in the synthesis of secretin by the repetitive excess mixed anhydride (REMA) method.

Protected secretin, a 27-peptide amide, was synthesized by the all-repetitive excess mixed anhydride (REMA) method and purified by preparative reverse-phase high-performance liquid chromatography. Highly potent secretin was obtained after deprotection with the aid of HF/anisole and purification by ion-exchange chromatography. The scope of the REMA-strategy is discussed in comparison with other strategies.