Ta-Min Chang

Neural Hormonal Regulation of Exocrine Pancreatic Secretion

Exocrine pancreatic secretion is regulated by hormone-hormonal and neural-hormonal interactions involving several regulatory peptides and neurotransmitter from the gut, the pancreas and the vagus nerve. The roles of the gastrointestinal peptides including secretin, CCK, neurotensin, motilin, PYY and pancreatic islet hormones including insulin, pancreatic polypeptide and somatostatin have been established. Interactions among secretin, CCK and neurotensin produce synergistic stimulatory effect. Motilin modulates the cyclic pattern of pancreatic secretion while local insulin provides a permissive role for the action of secretin and CCK at physiological concentration. Somatostatin, PYY and pancreatic polypeptide are inhibitory regulators, acting either on the release of secretin and CCK or on the action of the two stimulatory hormones. The vagal afferent-efferent pathway mediates the actions of many of these regulatory peptides, particularly of secretin and CCK. Acetylcholine and nitric oxide are the neurotransmitters known to mediate the actions of secretin and CCK. Serotonin (5-HT) released from enterochromaffin cells in the intestinal mucosa and nerve terminals of the enteric nervous system and intrapancreatic nerves may be involved in both stimulatory and inhibitory mechanism through its various receptor subtypes. 5-HT also mediates the action of secretin and CCK. The regulatory roles of neuropeptides, PACP and GRP, are now established, whereas those of others are being uncovered. Pancreatic juice provides both positive and negative feedback regulation of pancreatic secretion through mediation of both secretin- and CCK-releasing peptides. Three CCK-releasing peptides have been purified: monitor peptide from pancreatic juice, diazepam-binding inhibitor from porcine intestine, and luminal CCK-releasing factor from rat intestinal secretion. All have been shown to stimulate CCK release and pancreatic enzyme secretion. Pancreatic phospholipase A2 from pancreatic juice and intestinal secretion appears to function as a secretin-releasing peptide. However, the detailed map of neurohormonal regulatory pathways of exocrine pancreatic secretion is yet to be constructed.

Radioimmunoassay of cholecystokinin

A highly sensitive and specific radioimmunoassay for cholecystokinin (CCK) has been developed. Fully immunoreactive [125I]CCK33 was prepared by chloramine T-catalyzed iodination followed by purification by gel filtration and ion exchange chromatography. A high titer of antiserum was obtained by multiple immunizations of rabbits with 15% pure porcine CCK without conjugation. The antiserum was highly specific for CCK33 and CCK39, with 40% of the binding sites recognizing CCK8 at high affinity, but reacted weakly with gastrin. Plasma interference was eliminated by an XAD-2 resin column extraction technique with high recovery of CCK. The overall assay sensitivity was 3.3 pM with intra-and interassay coefficients of variation determined with a plasma of 11.2 pM at 9.6 and 20.8%, respectively. The assay was capable of detecting linear increments of both CCK33 and CCK8 added into plasma and intravenous infusion of CCK8 as low as 0.03 μg/kg/hr in dogs. The assay was validated by its ability to monitor increase of plasma CCK immunoreactivity after ingestion of a meat meal in both humans and dogs, as well as following intragastric infusion of liver extract meal and intraduodenal infusion of phenylalanine in dogs. When the CCK8 and gastrin binding sites of the antiserum were removed by immunoadsorption, the treated antiserum remained capable of measuring a postprandial change in plasma CCK concentration, indicating that CCK33-like immunoreactivity was present in the plasma.

Secretin, 100 years later.

One hundred years have elapsed since the discovery of secretin by Bayliss and Starling in 1902. In the past century, the research of secretin has gone by many milestones including isolation, purification and structural determination, chemical synthesis, establishment of its hormonal status by radioimmunoassay and immunoneutralization, identification of the specific receptor, cloning of secretin and its receptor, and identification of a secretin-releasing peptide. It has become clear that secretin is a hormone-regulating pancreatic exocrine secretion of fluid and bicarbonate, gastric acid secretion, and gastric motility. The release and actions of secretin is regulated by hormone–hormonal and neurohormonal interactions. The vagus nerve, particularly its afferent pathway, plays an essential role in the physiological actions of secretin. Substantial information about the property of the secretin receptor has been accumulated, but a potent secretin receptor-specific antagonist remains to be formulated. The neural regulatory mechanisms of the release and action of secretin await further elucidation. The physiological role of secretin in intestinal secretions and motility and extragastrointestinal organs remains to be defined. The presence of secretin and its receptor in the central nervous system is well documented, but its function as a neuropeptide has been recognized gradually and requires extensive study in the future.

Radioimmunoassay of secretin: A critical review and current status

The radioimmunoassay methods of secretin are reviewed with respect to production of antibody, preparation of radioactive tracers, and effect of plasma interference. The major difference in the secretin assay methods resides in handling plasma interference. Thus the assay sensitivity decreased markedly when the assay was conducted by diluting plasma samples. When the assay was conducted by compensating for plasma interference with homologous hormone-free plasma, the effect of plasma interference was greatly reduced, leading to a more sensitive assay. However, this method probably can not obtain consistent results with plasma samples collected under various experimental conditions. The method is still subject to considerable desensitization and assay variation. On the other hand, the elimination of plasma interference before assay results in the most sensitive secretin assays capable of detecting consistently a significant postprandial rise in plasma secretin level. It is concluded that a sensitive, validated secretin radioimmunoassay should be one that is capable of detecting increments of plasma secretin in response to doses of intraduodenal acid at 0.055 mEq/min or lower and intravenous administration of exogenous secretin at 0.03 CU/kg/hr with concomitant stimulation of pancreatic bicarbonate and water secretion. With a sensitive and accurate radioimmunoassay for secretin, it is now possible to further investigate the physiology and pathophysiology of secretin.

Mapping of motilin-immunoreactive Neurons of the rat brain

Motilin immunofluorescence was observed in the rat brain by means of N- and C-terminally directed antisera. A detailed mapping of the localization of motilin-like immunoreactive neurons in the rat forebrain is presented. Colchicine pretreatment revealed many cell bodies in the mediobasal hypothalamus. A small number of cells were observed in the organum vasculosum lamina terminalis (OVLT) region. A rich innervation of varicose fibers was observed in the median eminence and OVLT. Fibers of varying densities were observed in the preoptic area, nucleus interstitialis stria terminalis, hypothalamic nuclei, basal hypothalamus, amygdala, mammillary doby and central gray. The localization of these neurons suggests that motilin, like other brain peptides serves a variety of functions including neuroendocrine regulation.

Cholinergic role on release and action of motilin

In conscious dogs with gastric fistula and platinum electrodes on the antrum, duodenum and jejunum, IV atropine 100 micrograms/kg/hr and hexamethonium 10 mg/kg/hr, blocked cyclic increases in fasting plasma motilin concentration (PMC) and spontaneous migrating myoelectric complexes (MMCs) of both antrum and duodenum. The two drugs also blocked occurrence of premature MMCs produced by synthetic porcine motilin. In anesthetized dogs, electrical stimulation of cervical vagi with stimulation parameters: 9 V, 10 c/s, 5 msec, caused a significant increase in both portal and femoral venous PMC which was blocked by atropine. Fractionations of vagus nerve extracts by gel filtration using Sephadex G-50 superfine column revealed most of motilin-like immunoreactivity (MLI) with the same mobility as pure porcine motilin. Studies suggest that cholinergic influence plays a significant role on release of motilin.

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells.

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the sameM r as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.

Presence of biologically and immunologically active secretin-like substance in the mammalian brain

The present study involves the isolation and characterization of secretin-like immunoreactivity from the brains of pigs, rats and dogs. Secretin-like immunoreactivity was extracted with 0.1 N HCl and subjected to SP-Sephadex ion exchange chromatography and gel filtration on a Sephadex G-50 superfine column. The average amounts of secretin-like immunoreactivity in the extracts of 2 pigs, 7 rats and 6 dog brains were 0.25 ng/g, 2.4 +/- 0.2 ng/g and 0.34 +/- 0.07 ng/g fresh tissue weight, respectively. The secretin-like immunoreactivities in the brain extracts exhibited the same retention coefficient as natural porcine secretin on gel filtration and were eluted in the same salt gradient from the SP-Sephadex column. A partially purified secretin-like immunoreactivity isolated from canine brain exhibited the same bioactivity as natural porcine secretin to stimulate pancreatic volume flow in anesthetized rats (n = 4). These results indicated that secretin-like immunoreactivities from brain extracts possess the same molecular size and charge as natural porcine secretin and the secretin-like immunoreactivity isolated from dog brain is active in stimulating pancreatic secretion in anesthetized rats.

Plasma secretin concentrations and gastric pH in healthy subjects and patients with digestive diseases

Plasma secretin concentrations were determined in healthy subjects and patients with duodenal ulcer, achlorhydria, and celiac sprue. Mean fasting plasma secretin concentrations in 26 healthy subjects and 26 duodenal ulcer patients were 6.7±0.5 and 10.2±1.2 pg/ml, respectively, and were significantly different (P<0.02). After ingestion of a standard meat meal, pyloric pH decreased to less than 4.5 within 15 min and plasma secretin concentrations significantly increased in all 52 subjects. In 14 subjects (seven healthy subjects and seven patients with duodenal ulcer), no significant rise in plasma secretin concentration occurred when pyloric pH was maintained at greater than 5.0 by intravenous cimetidine (600 mg) and intragastric antacid. In 10 achlorhydric patients, intragastric pH remained greater than 5.0 after the meal and plasma secretin concentrations did not change. However, plasma secretin concentrations increased significantly when 0.1 N HCl was infused in the stomach (25 mEq/hr) during the postprandial period. In all eight adult patients with celiac disease (seven untreated, one partially treated), pyloric pH remained less than 4.0 after a meal. Postprandial secretin concentrations did not increase significantly in six and showed a transient rise in two. These studies show that (1) plasma secretin concentration increases significantly after meals in healthy subjects and patients with duodenal ulcer; (2) neutralization of gastric acid and the achlorhydric state show no significant postprandial rise in plasma secretin concentration; (3) achlorhydric patients do not have a defect in secretin release in response to acid; and (4) failure of postprandial rise in plasma secretin in patients with celiac disease is attributed to impaired release of secretin and in achlorhydric patients it is attributed to lack of acid secretion.

Release of cholecystokinin and exocrine pancreatic secretion in response to an elemental diet in human subjects.

We investigated in human volunteers the effects of an elemental diet (ED) containing amino acids on release of endogenous cholecystokinin (CCK) using a highly sensitive and specific radioimmunoassay of CCK and exocrine pancreatic secretion using a dye dilution technique with polyethylene glycol 4000 as a nonabsorbable marker. Intrajejunal administration of ED at three different infusion rates (12.5, 25, and 50 ml/30 min) resulted in a significant increase in plasma CCK concentration in a dose-related manner. Plasma concentrations of gastrin or secretin, however, did not change. Pancreatic secretion of protein, amylase, and bicarbonate also increased significantly. The change in pancreatic secretion of protein, amylase, and bicarbonate output paralleled that of the circulating CCK level but not that of plasma secretin. Thus, the dose of amino acid contained in ED recommended for clinical use can significantly stimulate the release of CCK from the upper small intestine, raising the plasma concentration of CCK. This level can evoke a significant increase in exocrine pancreatic secretion.