Secretin Molecule Research Articles

Magnetically labeled secretin retains receptor affinity to pancreas acinar cells.

Previously, we have developed a colloidal dextran-stabilized monocrystalline iron oxide nanocompound (MION-46) as a magnetic label for magnetic resonance imaging (MRI). In an effort to use this magnetic label to visualize pancreatic receptor function by MRI in vivo, we investigated the potential of secretin as a vector molecule. Secretin receptors, abundant on exocrine pancreas cells, recognize secretin through its amidated carboxyl terminal. In order to conjugate secretin to MION, we utilized the specific interaction between biotin and streptavidin, since direct conjugation of human secretin to MION has previously resulted in low yields and low affinity of the conjugate (unpublished results). Initially, we biotinylated the N-terminal primary amino group of secretin (60% yield). In a separate step, streptavidin (SA) was immobilized onto the surface dextran molecules of MION (79% yield) by reductive amination. Each secretin molecule was conjugated to one biotin molecule and each MION particle to an average of two SA molecules. The biotinylated secretin was then conjugated to MION through the biotin-streptavidin interaction (90% yield). The secretin-biotin-streptavidin-MION construct thus contained approximately two secretin molecules per MION. An in vitro competitive binding assay of pancreatic acinar cells demonstrated that the magnetically labeled secretin retained affinity to the secretin receptors. In vivo distribution studies in rats showed a significantly higher pancreatic accumulation of the secretin-biotin-streptavidin-MION construct as compared to the control group that had received unmodified MION. Our data indicate that bioactive peptides can be attached to dextran-coated iron oxide particles through the biotin-streptavidin interaction while retaining receptor affinity. Such target-specific agents have potential use in MR imaging to probe for a variety of receptor systems.

Differential effects of secretin‐fragments imply a dual mechanism of action for secretin

The effects of synthetic peptides, representing different parts of the secretin molecule in isolated mouse pancreatic islets have been investigated in perifusion studies. In the presence of 10 mM D-glucose the C-terminal nonapeptide Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2 (S19-27) showed a 2-fold higher activity than that earlier shown for S22-27 and had the same effect on the dynamic pattern of insulin release as secretin, while the elongating sequence Leu-Gln-Arg (S19-21) had no effect on the insulin release. The nonapeptide Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg (S10-18) had no influence on the insulin release. Glucagon release seen after intact secretin could not be shown for any of the smaller fragments. Accumulation of cAMP in the islets as seen with secretin, could at 10 mmol/L D-glucose only be demonstrated with S22-27 or S19-27 but not with S10-18 or S1-6. Our results indicate that full size secretin has to be present to stimulate glucagon release while insulin-releasing activity can be confined to the C-terminal part of the hormone.

Effect of dose, pH, and osmolarity on nasal absorption of secretin in rats. III. In vitro membrane permeation test and determination of apparent partition coefficient of secretion.

Nasal absorption of secretin in rats was enhanced in an acid solution and the maximum absorption was observed at a sodium chloride solution molarity of 0.462. In order to predict how changes in the secretin molecule would affect its absorption through the nasal mucosa independently of structural changes in the epithelial membrane, an artificial membrane permeation test was conducted, and the apparent partition coefficient between octanol and a test solution was determined. The concentration of secretin was measured using high performance liquid chromatography. The amount of secretin that permeated through an artificial membrane was hardly affected by changes in pH, which suggest that the size of the secretin molecule was not changed. The apparent partition coefficient, however, increased as the pH of the test solution rose from 3.81 to 7.0, which suggested that the hydrophobicity of secretin was enhanced. In relation to the osmolarity of the test solution, the amount of permeation was hardly affected by the concentration of sodium chloride, but the partition coefficient increased with the concentration of the sodium chloride solution. It was supposed that the size of the secretin molecule was not changed in spite of the increasing hydrophobicity, and the nasal absorption of secretin at a sodium chloride molarity of 0.462 was dependent on a change in the epithelial cells. When sorbitol was used as an osmoregulatory agent, the apparent partition coefficient hardly varied as the osmolarity of the solution was increased, whereas the amount of permeation decreased, and these findings reflected the nasal absorption in rats.

Secretin and its C-terminal hexapeptide potentiates insulin release in mouse islets.

Peptides representing the C-terminal end of secretin were synthetized and their effects tested along with secretin on column-perifused isolated mouse pancreatic islets. Insulin release induced by 10 mmol/l D-glucose was potentiated by secretin tested in a concentration range of 0.01-10 micrograms/ml; the maximal effect was obtained with 1 microgram/ml secretin. This effect was mimicked by 50-500 micrograms/ml NH2-Leu-Leu-Gln-Gly-Leu-Val-NH2, [S-(22-27)], which represents an amidated C-terminal sequence of the secretin molecule. The consecutive smaller secretin C-terminal peptides had either no effects [Val-NH2, S-(24-27)] or only marginally [S-(26-27), S-(23-27)] potentiating effects on insulin release in the presence of 10 mmol/l D-glucose. The effects of secretin and S-(22-27) were not influenced by 2 mmol/l glutamine. The intact hormone and the five synthetic peptides as well as Val-NH2 had no stimulatory effect on islet glutamate dehydrogenase activity. In fact, S-(23-27), S-(24-27), and S-(25-27) inhibited the islet glutamate dehydrogenase activity, the activation by which amino acids and amino acid derivatives are known to elicit a potentiation of insulin release. Our results suggest that the C-terminal part is important to the marked potentiation of glucose-induced insulin release in vitro by secretin.

Conformation of peptides of the secretin-VIP-glucagon family in solution

The significance of a well defined molecular architecture in hormone receptor interaction and the methods available for the study of preferred conformations are discussed. The conformational freedom in glucagon is a major obstacle in the determination of its biologically relevant geometry. In the secretin molecule intramolecular forces generate a folded, partially helical conformation. In respect of long range cooperative interactions resulting in a compact molecule with secondary-tertiary structure secretin is similar to globular proteins. In VIP some characteristics of secretin and also of glucagon can be recognized. Further progress in conformation analysis can be expected from the study of rigid, cyclic analogs in which the biological activities of the parent hormones are retained or even enhanced. Such analogs have well defined conformations without external stabilization from membrane mimicking lipids. Therefore, they provide information on the biologically relevant geometry of the hormones and contribute also to our knowledge of receptor sites.

Conformation of peptides of the secretin-VIP-glucagon family in solution

The significance of a well defined molecular architecture in hormone receptor interaction and the methods available for the study of preferred conformations are discussed. The conformational freedom in glucagon is a major obstacle in the determination of its biologically relevant geometry. In the secretin molecule intramolecular forces generate a folded, partially helical conformation. In respect of long range cooperative interactions resulting in a compact molecule with secondary-tertiary structure secretin is similar to globular proteins. In VIP some characteristics of secretin and also of glucagon can be recognized. Further progress in conformation analysis can be expected from the study of rigid, cyclic analogs in which the biological activities of the parent hormones are retained or even enhanced. Such analogs have well defined conformations without external stabilization from membrane mimicking lipids. Therefore, they provide information on the biologically relevant geometry of the hormones and contribute also to our knowledge of receptor sites.

Production in Escherichia coli of biologically active secretin, a gastrointestinal hormone.

A synthetic gene for porcine secretin was ligated with Pst I-cleaved pBR322 by its flanking synthetic Pst I linkers to produce a fused protein consisting of an amino-terminal portion of beta-lactamase and an entire secretin molecule. The hybrid plasmid was transferred into competent Escherichia coli cells. The plasmids, which were proved in our investigation to contain the secretin gene in the desired orientation, were then screened for the production of secretin. This was shown by radioimmunoassay and gel electrophoretic analysis of the polypeptides that were synthesized in E. coli minicells transformed with the hybrid plasmids. Secretin so produced, whose carboxy terminal residue may not be amidated in contrast with natural porcine secretin, showed the same activity in stimulating pancreatic secretion in a bioassay with anesthetized rats as does natural secretin.

In vivo effects of five secretin analogs on fluid and potassium secretion from the rat pancreas

The importance of the N-terminal part of the secretin molecule for inducing fluid and potassium secretion from the pancreas was tested on anesthetized rats by comparing the biological capacity of bolus intravenous injections of secretin, secretin analogs, and the secretin (7–27) fragment. Except in one case, the relative potencies with which these peptides influenced fluid secretion correlated with the potencies on potassium secretion. [Glu 3]secretin and [Asn 3]secretin were 2–3 and 14 times less potent, respectively, than secretin. [Ala 4]secretin, [D-Ala 4]secretin and secretin were almost equipotent. [Val 5]secretin was as potent as secretin on water secretion but 2-fold less potent on potassium secretion. Secretin (7–27) was at best a very weak agonist of secretin.

Adsorption of secretin on glass surfaces.

Adsorption of secretin on a silicone-coated glass surface was compared with that onto a non-coated surface using controlled pore glass beads. The silicone-coated glass beads adsorbed 20 times more secretin than the non-coated beads. The adsorption was slightly affected by pH and amino acids, but was reduced to an insignificant amount in the presence of bovine serum albumin. It is suggested that the preferential adsorption of this peptide hormone onto a silicone-coated glass surface is probably the result of hydrophobic attraction between non-polar moieties of the secretin molecule and non-polar portions of the silicone membrane.

The interaction of secretin with pancreatic membranes

1. 1. 125I-labelled secretin bound rapidly and specifically to membranes from cat pancreas. Binding of labelled hormone was competitively inhibited by unlabelled secretin in the same range of concentrations that stimulated pancreatic adenylate cyclase in these membranes. The dissociation constant of the membrane binding sites for unlabelled secretin as evaluated by these displacement experiments was 4.1 · 10 −9 M and the number of binding sites 1.0 pmol per mg of membrane protein. 2. 2. Studies using different concentrations of [ 125I]secretin (at a constant ratio of labelled to unlabelled hormone) revealed a similar value of 4.4 · 10 −9 M for the dissociation constant. 3. 3. Both the association and dissociation rate constants of [ 125I[secretin binding were temperature sensitive; the dissociation rate constant increased more rapidly with increase in temperature. The ratio k −1/k +1 (at 22 °C) gave a dissociation constant of 3.7 · 10 −9 M which agrees closely with the figure obtained from equilibrium data. These data indicate that 125I-labelled secretin and unlabelled secretin bind to the same binding site on pancreatic membranes, with high affinity. 4. 4. Unlabelled secretin stimulated pancreatic adenylate cyclase with an apparent K m of 8.4 · 10 −9 M, while [ 125l]secretin apparently did not stimulate the adenylate cyclase. Together with the binding data this might suggest that different portions of the secretin molecule are responsible for binding and adenylate cyclase activation. 5. 5. Studies on the specificity of [ 125l]secretin binding carried out with various peptide hormones (glucagon, human gastrin, pancreozymin and caerulein) which are all inefficient in stimulating pancreatic fluid secretion, showed that these hormones have no influence on the binding of [ 125I]secretin. In contrast, vasoactive intestinal polypeptide, which stimulates pancreatic fluid and bicarbonate secretion, showed a competitive inhibition of secretin binding to the plasma membrane preparation.

Superior immunoreactivity of 125I (Des-Tyr-betaAla)-secretin with rabbit anti-secretin sera compared to 125I-secretin and 125 I-6-Tyrosyl secretin.

A secretin analogue in which the normal amino acid sequence had been elongated by a (Des-Tyr-betaAla)-residue was studied as tracer for secretin radioimmunoassay. 125I-(DATA)-secretin exhibited superior immunoreactivity with several rabbit anti-secretin sera compared to 125I-6-Tyr-secretin and also to secretin iodinated at its N-terminal histidyl residue. This may be due, at least in part, to higher conformational integrity of the secretin moiety in the 125I-(DATA)-secretin molecule. Thus, at present, 125I-(DATA)-secretin appears to be most suitable as tracer for sensitive secretin radioimmunoassay.

Preparation and specificity of antiserum to synthetic secretin and its use in a radioimmunoassay (RIA).

Secretin antibodies were produced in rabbits by immunization with synthetic secretin coupled to bovine serum albumin. An antiserum was obtained which reacted with similar strength with the complete secretin molecule and the C terminal tricosapeptide (residues 5 through 27) to a lesser extent (50%) with the C terminal tetradecapeptide (residues 14 through 27) and only minimally (0.03%) with the N—terminal tetradecapeptide (residues 1 through 14). Insulin, human growth hormone, ACTH, FSH, LH, glucagon and gastrin I did not react with the antibody. Natural cholecystokinin—pancreozymin crossreacted weakly (0.03%) probably as a result of contamination with secretin. Using this antiserum and 125I—labeled secretin of high specific activity a sensitive and specific radioimmunoassay for the measurement of secretin has been developed. Sensitivity of the assay was 25 pg. Coefficients of variation of intra and interassay reproducibility were 9.4% and 17.1%, respectively. Mean fasting serum immunoreactive secretin (IR...

Lipolytic activity of a peptide fragment of porcine secretin.

Synthetic porcine secretin, a peptide chain of 27 amino acids, accelerates li poly sis in rat and mouse fat cells. Six synthetic fragments of the porcine secretin molecule have now been assayed for lipolytic activity on free fat cells of the rat: peptides 23–27, 14–27, 9–27, 5–27, 5–14 and 1–14. Only peptide 1–14 is active; its minimal effective dose, 1 μg/ml, is 100 × greater on a weight basis and 200 × greater on amolar basis than that of secretin itself in the same assay system. It is concluded that the lipolytic property of secretin resides in the NH2- terminal half of the hormone molecule, 1–14; that sequence 1–4 plays an essential role in the activity of 1–14; and that 15–27, either in whole or in part, enhances the effect of the active region 1–14. (Endocrinology 85: 610,1969)

Effect of Secretin on Acid and Pepsin Secretion in Cat and Dog

Secretin in doses which produced a maximal inhibition of gastric secretion in the dog and maximal stimulation of pancreatic secretion in the cat had no effect on gastrin-stimulated gastric secretion in the cat. Irrigation of the duodenum with acid produced a much smaller inhibition of gastrin-stimulated gastric secretion in the cat than in the dog, thus suggesting that endogenous secretin is not so important for the control of acid secretion in the cat as in the dog. We conclude that the disparity between the action of secretin on gastric secretion in the cat and dog represents a true species difference. Identical doses (on a body weight basis) of secretin, however, produced strong stimulation of pepsin secretion in both the cat and dog. Stimulation was produced by continuous intravenous infusions of secretin in doses submaximal for pancreatic secretion. In addition the effect of natural secretin was reproduced exactly by the synthetic hormone, indicating that the stimulation of pepsin secretion is a property of the secretin molecule. Endogenous secretin, released by either exogenous acid in the duodenum or by closing the gastric fistula, reproduced the effect of the exogenous secretin or pepsin secretion. We conclude, therefore, that the stimulation of pepsin secretion is a physiological action of secretin.