Secretin Preparations Research Articles

Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin.

Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.

Preparation of 125I-labeled synthetic porcine secretin for radioimmunoassay.

Synthetic porcine secretion was labelled by the conjugation-labelling method of Bolton & Hunter, the lactoperoxidase method, the gaseous diffusion method, and the chloramine-T method. The chloramine-T technique was adapted as routine method. Ten mug (3.27 nmol) peptide was reacted with 5 mCi of Na125I at a concentration of chloramine-T of 1.3 mmol/l. Synthetic secretin was suitable for labelling for at least eight months when stored as dry matter in nitrogen-filled glass ampoules. Purification and separation of labelled from unlabelled hormone was carried out by gel-permeation chromatography on Sephadex G-50 superfine. The labelled preparation had a specific radioactivity of 405 +/- 33 muCi/nmol (mean +/- SEM., n = 9) and was unable for six days. 6-tyrosyl-secretin took more iodine compared to porcine synthetic secretin but had lower immunoreactivity with all antisera tested.

Preparation of 125I-labeled secretin of high specific radioactivity

A simple and rapid method of preparing 125I-labeled secretin of high specific radioactivity has been developed. Synthetic porcine secretin was iodinated with Na[ 125I] by a modification of the chloramine T method. Purification and separation of labeled from unlabeled secretin was achieved by chromatography on SP-Sephadex C-25 column. The labeled secretin possessed specific radioactivity as high as 500–550 μCi/μg.

The effect of natural and synthetic secretin on insulin secretion (author's transl)

The effect of natural secretin (G.I.H.) and synthetic secretin (Wünsch) on the secretion of insulin was investigated. Natural as well as synthetic secretin causes a short uniphasic release of insulin without influence on the concentration of glucose in the serum. Since the insulin releasing effect on synthetic secretin was demonstrated, it is clear, that the release of insulin by preparations of natural secretin mentioned in medical literature is not due to contamination by other peptides.

The effect of aminophylline on insulin release induced by secretin and cholecystokinin-pancreozymin in normal humans.

ABSTRACT The purpose of this study is to search the possibility that insulin-secretion, induced by the administration of pharmacological doses of relatively purified preparations of secretin and cholecystokinin–pancreozymin (CCK-PZ), might be mediated through the activation of the adenyl-cyclase system in the β-cell of normal humans. To test this hypothesis the effect of a 15-min infusion of 250 mg of aminophylline on insulin release induced by cholecystokinin-pancreozymin (CCK-PZ) and secretin was studied in 12 normal subjects. CCK-PZ (2 U/kg/body wt) iv administered to 6 subjects provoked a quick and transient insulin release. In a second test aminophylline (250 mg) was infused for 15 min before CCK-PZ (2U/kg/body wt) was given; in this test a significantly enhanced insulin secretion was observed in every subject. The effect of secretin was tested in an identical way in 6 subjects. It was observed that the infusion of aminophylline previous to the secretin injection (2 U/kg/body wt) elicited a significa...

Adenylate cyclase in the rat pancreas properties and stimulation by hormones

1. 1. In view of evidence for a role of cyclic AMP in the secretin-stimulated water and electrolyte secretion and the pancreozymin-stimulated enzyme secretion by the pancreas, the occurrence of adenylate cyclase in the pancreas has been investigated. 2. 2. The presence of the enzyme in a particulate fraction of rat pancreas has been demonstrated. Basal activity is very low under standard conditions (less than 3 pmoles/min per mg protein), but 10 mM NaF stimulates the enzyme to 15–40 pmoles/min per mg protein. 3. 3. Properties of the enzyme have been determined in the presence of 10 mM NaF. The apparent K m for ATP, derived from a Lineweaver-Burk plot is 0.3 mM. At ATP concentrations above 1.5 mM there is substrate inhibition. The optimal pH is 7.4. The enzyme requires Mg 2+ for its activity, concentrations of 5 mM and higher giving maximal activity. Ca 2+ in concentrations above 0.1 mM inhibits the enzyme. 4. 4. The enzyme is stimulated by a synthetic secretin preparation (half maximally activating concentration 1.5·10 −8 M) as well as by a purified pancreozymin preparation (half maximally activating concentration 1.5·10 −6 M). The latter activation is not due to a contamination of the pancreozymin preparation by secretin. The secretin-stimulated enzyme has the same optimal pH of 7.4 and shows the same Ca 2+ inhibition as the NaF-stimulated enzyme. 5. 5. The enzyme is not stimulated by adrenalin and its analogues, by acetylcholine and its analogues, and by the hormones glucagon and gastrin (synthetic). 6. 6. Specific enzyme activity (per mg protein) in enzyme preparations obtained from rats with free access to food and stimulated in vivo by secretin and pancreozymin is twice as high as that in enzyme preparations from starved animals. 7. 7. The physiological significance of these findings is discussed.

Relationships between blood flow and secretion in the isolated perfused canine pancreas.

1. Stimulation by a continuous intra-arterial infusion of secretin caused a marked vasodilatation in the pancreato-duodenal preparation or in the isolated pancreas perfused with whole heparinized blood. There was no difference in the vasomotor effects of two unequally purified preparations of secretin. The fact that the vasodilatation was still observed when the duodenum was removed proved that the haemodynamic effect of secretin was initiated in the pancreas itself.2. The secretory response of the pancreas to a given dose of secretin was larger when the blood pressure was kept constant by increasing the blood flow than when the blood pressure was allowed to fall by maintaining the blood flow at a fixed value. It is concluded that the vasodilatation plays a role in the functional regulation of the pancreatic response to secretin.3. The correlation between blood flow and arterial pressure on the one hand, and the secretory response to secretin on the other, was not mediated by the control of respiration since there was no variation in oxygen uptake in response to a variation in the blood flow. This correlation was still observed when the venous blood was discarded and therefore could not be explained by variations in the rate of recirculation of secretin. It was observed too in experiments where the duodenum was removed, the pancreas alone being introduced into the perfusion apparatus, which indicated that this effect was not mediated by a control of the release of secretin by the duodenum.

Glucagon stimulates Brunner's gland secretion.

GLUCAGON and secretin, chemical homologues having 14 similar amino-acids in identical positions1, induce choleresis2, stimulate insulin release3 and inhibit gastric secretion4. Glucagon, however, inhibits, while secretin stimulates, pancreatic fluid and electrolyte secretion5. Because three different preparations of secretin—synthetic porcine (Squibb), pure natural porcine (Jorpes and Mutt) and impure porcine (Boots)—stimulate Brunner's gland secretion6–8, we investigated whether glucagon would affect Brunner's gland secretion in dogs.

Studies on the inactivation of pancreozymin concentrates.

ABSTRACT The loss in hormonal activity resulting on incubation of pancreozymin concentrates with canine blood serum at 37°C has been investigated. The test extracts comprised (SI), a solid obtained from treatment of a salted acid extract of the upper intestinal mucosa with trichloroacetic acid and the amine precipitate, (AP), derived from SI. The decrease in activity was determined in relation to an incubated concentrate-saline solution in dogs with the main pancreatic duct cannulated. Fluid secretion was promoted by potent secretin preparations in saline administered by single injection or by continuous infusion. As based on the total amylase content of the pancreatic juice samples, the hormonal concentrates underwent rather small losses in activity, the AP requiring higher dilutions with serum for an effect as compared to SI. Canine liver extract and a hog kidney diamine oxidase concentrate caused inactivation of pancreozymin products but bovine and canine plasma protein fractions, heavy metal salt and thiol solutions and possibly, human urine, were without action.

The effect of intestinal extracts of lampreys ( Lampetra fluviatilis and Petromyzon marinus) on pancreatic secretion in the rat

Using a procedure based on the method of Friedman and Thomas for the preparation of secretin and pancreozymin, extracts have been made of the intestine of the river lamprey ( Lampetra fluviatilis) and the marine lamprey ( Petromyzon marinus), trapped during their anadromous spawning migration when feeding has ceased. It is shown, using the assay procedure of Love, that methanol-soluble and methanol-insoluble fractions from both species evoke in the rat an increased flow of pancreatic secretion and an increase in its protein concentration. Some evolutionary implications of the results are briefly discussed, but the need for further analysis of the situation is emphasized.

The effect of synthetic secretin on the external pancreatic secretion in dogs.

Henriksen, F. W. The effect of synthetic secretin on the external pancreatic secretion in dogs. Acta physiol. scand. 1968. 72. 433–440. The effect of synthetic secretin on the external pancreatic secretion was investigated in three dogs with Thomas fistulas and gastrostomies. The dose-response curve was determined in two dogs. The maximal secretory capacity equalled that obtained with other secretin preparations. Supramaximal doses of synthetic secretin did not depress the secretion of fluid or bicarbonate. A short-lasting increase in the protein secretion after injection of synthetic secretin is explained as a “wash-out” phenomenon.

The Effect of Synthetic Secretin on the External Pancreatic Secretion in Dogs

AbstractHenriksen, F. W. The effect of synthetic secretin on the external pancreatic secretion in dogs. Acta physiol. scand. 1968. 72. 433–440.The effect of synthetic secretin on the external pancreatic secretion was investigated in three dogs with Thomas fistulas and gastrostomies. The dose‐response curve was determined in two dogs. The maximal secretory capacity equalled that obtained with other secretin preparations. Supramaximal doses of synthetic secretin did not depress the secretion of fluid or bicarbonate. A short‐lasting increase in the protein secretion after injection of synthetic secretin is explained as a “wash‐out” phenomenon.

The effects of secretin, pancreozymin, and gastrin on insulin and glucagon secretion in anesthetized dogs.

The effects upon islet hormone secretion of highly purified preparations of secretin and of pancreozymin-cholecystokinin and of a crude gastrin-containing extract of hog antrum have been studied in acutely operated dogs. All three preparations were shown to cause a striking increase in insulin concentration in the pancreaticoduodenal venous plasma after their rapid endoportal injection in anesthetized dogs. With each hormone preparation, the peak in insulin secretion occurred 1 minute after injection, and a rapid decline was observed immediately thereafter. Whereas secretin and gastrin failed to alter significantly the pancreaticoduodenal venous glucagon or arterial glucose concentration, pancreozymin caused a dramatic rise in pancreaticoduodenal venous glucagon concentration, which reached a peak 3 minutes after injection, and hyperglycemia was noted to occur soon thereafter. Endoportal infusion of secretin and pancreozymin for 20 minutes caused responses that were sustained but qualitatively identical to the responses noted after rapid injection of the hormones. The beta-cytotropic effect of secretin was abolished by the infusion of epinephrine. These results could not be attributed to the small degree of contamination of the enteric hormone preparations with insulin or glucagon, and it would appear that secretin, pancreozymin, and probably gastrin have insulin-releasing activity and that pancreozymin has, in addition, glucagon-releasing activity.The demonstration that these three hormones possess insulin-releasing activity suggests that there is in the gastrointestinal tract a chain of betacytotropic hormones from antrum to ileum that is capable of augmenting insulin secretion as required for disposal of substrate loads. It is suggested that the existence of this "entero-insular axis" prevents high substrate concentrations that would otherwise follow ingestion of large meals were the insular response entirely a function of arterial substrate concentration.

The effect on pancreatic flow of acetylated secretin preparations

Summary Secretin was treated with acetic anhydride and the resulting preparation (Sa) was tested in dogs with Thomas duodenal cannulas for its effect on pancreatic flow. Sa caused pancreatic secretion when given as a constant infusion or as a single intravenous injection. A single injection of a large dose of Sa was apparently less stimulatory than a comparable amount of secretin. When a large dose of Sa in the form of a single injection was superimposed on a constant infusion of either secretin or Sa, there was a significant suppression of pancreatic flow. It is postulated that acetylated secretin competes with secretin for receptor sites in the pancreas. The failure to suppress pancreatic flow completely may be related to deacetylation or to residual secretory activity of the acetylated material.

On the biological assay of secretin. The reference standard.

AbstractThe authors discuss the methods in use for the assay of secretin preparations in dogs, cats and rats. A detailed description is given of the method of Mutt and Söderberg, maintaining the cats anesthetized continuously for 4–5 days. Particular attention is paid to recent methods using rats for the secretin assay. The numerical values of the different secretin units, suggested by different authors, are discussed. The difficulties encountered in selecting a reference standard are discussed. The authors recommend frequent controls of the strength of the standard with any of the familiar techniques.

Comparison of subcutaneous and intravenous administration of pancreatic stimulants.

In conscious dogs with permanent gastric and pancreatic fistulas, the response to single injections of extracts of mucosa of the gastrointestinal tract by intravenous and subcutaneous routes was compared. Both crude and highly purified secretin preparations were weak stimulants of the pancreas by subcutaneous administration, in contrast to the large effect of the same doses given intravenously. An extract containing pancreozymin was effective in causing increased protein secretion by the pancreas by both routes of injection. Gastrin stimulated pancreatic volume flow by subcutaneous administration but not by intravenous administration, in contrast to secretin. This indicates that crude extracts of the mucosa of the pyloric gland area of the hog do not contain enough secretin to be easily detectable. Attempts to show that supramaximal doses of secretin, pancreozymin, and gastrin exhibit self-inhibitory properties when tested on pancreatic secretion were unsuccessful, although self-inhibition of gastrin-stimulated gastric secretion was confirmed.

Stimulation of Pancreatic Secretion by Extracts of the Pyloric Gland Area of the Stomach

Summary In dogs with a new type of chronic pancreatic fistula and a gastric fistula, extracts of mucosa of the pyloric gland area of the stomach of hogs (gastrin extracts) have been shown to stimulate pancreatic as well as gastric secretion. The effect on protein output by the pancreas was particularly marked; that on volume flow and bicarbonate secretion less so. Dose-response curves for gastric and pancreatic stimulation by gastrin extracts, commercial preparations of secretin and pancreozymin, and extracts of hog fundic and intestinal mucosa have been compared.

THE INFLUENCE OF CHEMICALLY PURE SECRETIN ON HEPATIC BILE OUTPUT.

AbstractJonson, G., L. Sundman, and L. Thulin. The influnce of chemically pure secretin on hepatic bile output. Acta physiol. scand. 1964. 62. 287–290. — In awake cholecystectomized dogs with a permanent external common duct fistula the bile output increased during infusion of a secretin preparation, containing about 20,000 clin. units per mg of substance (Jorpes and Mutt 1961). At the largest dose, about 43 clin. units per kg b. wt. and hour, it was about 3 times the initial output, and even then it was smaller than the output during digestion of food with bile. The relation between the log of the secretin dose and the bile output was linear.

COMPARISON OF GASTRIC INHIBITORY PROPERTIES OF TWO SECRETIN PREPARATIONS.

Commercial preparations of secretin inhibit gastric secretion of Heidenhain pouches in response to certain stimuli. Since these preparations are mucosal extracts and not pure secretin, inhibition could be due to active materials other than secretin present in the extracts. To determine whether other active substances were present, two commercial secretin preparations, which were markedly different in weight per unit of pancreatic secretagogic activity, were tested for their relative abilities to stimulate pancreatic secretion in dogs with Thomas cannulas and to inhibit gastric secretion of Heidenhain pouches stimulated by exogenous gastrin in totally gastrectomized dogs. Inhibition of gastric secretion occurred with both preparations. The ratio between the weight of extract required to inhibit gastric secretion and that required to stimulate pancreatic secretion was the same for the two preparations despite the great differences in weights necessary to produce equivalent stimulation of the pancreas. This supports the concept that the same factor, either secretin or a closely related substance, accounted for both of these effects.

Effect of secretin given into portal or peripheral vein.

Intravenously injected secretin is considered generally to equal the physiological effect of the hormone as liberated during digestion. However, there is reason to assume a difference in effect on the pancreas between these 2 ways of entrance of the hormone into the circulation, because a number of hormones seem to be inactivated in the liver. The naturally secreted secretin most probably enters the portal circulation and passes through the liver first, while in intravenous injection it passes the liver only after it has been diluted in the general circulation (1). This concept is supported by results obtained with vivdialysis by Necheles and Lim (2). Previous workers, using rather crude preparations of secretin, found a weaker response of pancreatic secretion following its injection into the portal vein than into a systemic vein(3). However, Mellanby(1) using a more purified secretin, found no such difference. Our attention was focused on this question when we were able to obtain a highly purified secret...