Primary Pancreatic Secretinoma: Further Evidence Supporting Secretin as a Diarrheogenic Hormone.

OBJECTIVES: To document the existence of primary pancreatic secretinoma in patients with watery diarrhea syndrome (WDS) and achlorhydria and establish secretin as a diarrheogenic hormone. BACKGROUND: Vasoactive intestinal peptide (VIP) has been widely accepted as the main mediator of WDS. However, in 1968, Zollinger et al reported 2 female patients with pancreatic neuroendocrine tumors, WDS, and achlorhydria. During surgery on the first, a 24-year-old patient, they noticed distended duodenum filled with fluid and a dilated gallbladder containing dilute bile with high bicarbonate concentration. After excision of the tumor, WDS ceased and gastric acid secretion returned. The second, a 47-year-old, patient's metastatic tumor extract given intravenously in dogs, produced significantly increased pancreatic and biliary fluid rich in bicarbonate. They suggested a secretin-like hormone of islet cell origin explains WDS and achlorhydria. These observations, however, predated radioimmunoassay, immunohistochemical staining, and other molecular studies. METHODS: The first patient's tumor tissue was investigated for secretin and VIP. Using both immunohistochemistry and laser microdissection and pressure catapulting technique for RNA isolation and subsequent reverse transcription polymerase chain reaction, the expression levels of secretin, and VIP were measured. RESULTS: Immunoreactive secretin and its mRNA were predominantly found in the tumor tissue whereas VIP and its mRNA were scarce. CONCLUSIONS: The findings strongly support that the WDS and achlorhydria in this patient may have been caused by secretin as originally proposed in 1968 and that secretin may act as a diarrheogenic hormone.

Endocrine cells in the human common bile duct in patients with obstructive jaundice.

BACKGROUND/AIMS: There was no data about endocrine cells in the extrahepatic bile duct in secondary cholangitis due to obstructive jaundice. The aim of the present study is to investigate immunohistochemically the endocrine cell types in the lower part of the human common bile duct in biopsy samples, collected during drainage because of complete or incomplete obstruction, caused mainly by stones. We explained the presence of various hormone-producing endocrine cells in this region with the regulation of physiological and pathological processes there. METHODOLOGY: We used light and electron microscopic immunohistochemistry. RESULTS: More gastrin-positive, somatostatin-positive, secretin-positive, serotonin-positive, chromogranin- A-positive and synaptophysin-positive endocrine cells were found compared to control preparations. CONCLUSIONS: Occurrence of endocrine cells may relate to disturbed bile flow and to formation of calculi. Endocrine cell hyperplasia may be related to longstanding inflammation as in chronic cholecystitis and all secreted hormones from the described ECs can support pathologic process in the choledochus, i.e. inflammation, increased mucus secretion, fibrosis, muscle contraction, etc. We may state that various ECs (similar to those in duodenum) present in the lower part of the large bile duct and their hormones exert action on physiology (motility, secretion) and pathology (inflammation and fibrosis) in that part of the biliary tree.

Cholangiocyte anion exchange and biliary bicarbonate excretion.

Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. The excretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.

[Clinicopharmacological study of gastrointestinal drugs from the viewpoint of postmarketing development].

Pharmaceutical development starts with the discovery of a new compound. Drugs become commercially available after non-clinical and clinical studies, but processes that take place after marketing are also important for pharmaceutical development. In recent years, use of the phrase "Ikuyaku" meaning postmarketing development has become more common. Sometimes, the proper usage, indications and harmful effects of a drug are discovered only after it becomes commercially available and is administered to many patients. Hence, pharmacists need to actively perform postmarketing studies to reveal the true nature of drugs. In the present clinicopharmacological study, we investigated the effects of histamine H(2) receptor antagonists (H(2)-RAs) on the plasma concentrations of gastrointestinal peptides from the viewpoint of postmarketing development. First we established an enzyme immunoassay for secretin, which is involved in gastrointestinal motility. Then we used this and existing peptide assays to investigate the above-mentioned issues. Ranitidine and nizatidine increased the plasma concentration of motilin. It is believed that the plasma concentration of Ach is elevated by ranitidine and nizatidine, which possesses an anti-AchE activity, and that the increased the plasma concentration of Ach facilitated release of motilin, elevating the plasma concentration of motilin. When compared to the placebo, lafutidine significantly increased the plasma concentration of CGRP (calcitonin gene-related peptide) and substance P. Furthermore, released CGRP stimulated CGRP1 receptors to facilitate secretion of somatostatin. Therefore, lafutidine appears to protect the gastric mucosa and regulate gastrointestinal motility. The same results were obtained with ranitidine and nizatidine. While H(2)-RAs have a common function in suppressing the secretion of gastric acid, they do not exhibit the same effects on factors related to recurrence of peptic ulcer, such as gastrointestinal motility and blood flow in the gastrointestinal mucosa. Hence, measuring the plasma concentration of gastrointestinal peptides can be used to estimate the effects of drugs on gastrointestinal motility. From the viewpoint of postmarketing development, we are in the process of establishing indicators for the proper usage of pharmaceutical drugs. Pharmacists need to closely follow and monitor adverse reactions. In order to further improve monitoring of drug therapy, it will be necessary to assess not only the blood concentrations of drugs, but also biological reactions to the drugs. Since the levels of peptides reflect the clinical efficacy of gastrointestinal drugs, measuring peptide levels appears to be useful for selecting appropriate drugs.

Brain/gut peptides in fed and fasted rats.

The concentrations and contents of vasoactive intestinal peptide (VIP) and cholecystokinin (CCK) in the brain and of these peptides along with secretin and glucagon-like immunoreactivity (GLI) in the gut were compared in a group of 16 5-day fasted adult Sprague-Dawley rats with the corresponding peptides in a group of 16 nonfasted littermates. The mean weight of the fasted rats at the beginning of the study was 263 +/- 10 g (+/- SEM) and was 177 +/- 7 g before killing, for a net loss of 33% of initial body weight; the 16 fed rats increased their mean weight from 225 +/- 11 to 284 +/- 12 g, for a net gain of 12%. During the 5-day fast there was no change in the weight of the cortex, hypothalamus, or brain stem. However, the weight of tissues from the gut decreased to about half the weight of the corresponding tissues in the fed animals. There was no significant change in brain VIP or CCK. VIP content in the gut was unchanged. However, because of the decrease in organ weight, its concentration almost doubled. Secretin concentrations in the gut of fasted rats did not change significantly, but organ contents fell to about half. The gut content of GLI also fell by half or more. The concentrations of CCK in methanol extracts of the duodenum and jejunum remained relatively constant, but those in acid extracts fell by 40% in the fasted animals. This represents an approximately 70% decrease in organ content of CCK. These findings are interpretable as demonstrating that during a prolonged fast neuronal CCK and VIP are well conserved, but endocrine CCK, secretin, and GLI are markedly decreased because of loss of intestinal mucosa.

Secretin-like immunoreactivity and biological activity in the antral mucosa.

Using immunohistocytochemical techniques, secretin cells are again demonstrated in the antral mucosae of both dogs and rats. Secretin-like immunoreactivity was found in the crude extracts of antral mucosae in 15 dogs [1.18 +/- 0.48 (+/- SE) ng/g wet wt of mucosae], and a similar amount of SLI was also found in 82 rat antral mucosae. Upon ion exchange chromatography, the extracts of dog antral mucosae exhibited a predominant species eluted by the same salt concentration as porcine secretin. The rat antral mucosal extract also produced a chromatogram exhibiting the same predominant species on the ion exchanger. The main immunoreactive secretin peak, when gel filtrated on a Sephadex G-50 (superfine) column, produced an elution profile identical to that of standard natural porcine secretin. These results indicated that antral mucosae of both animal species contain an immunoreactive secretin-like material of the same charge and size as natural porcine secretin. Intravenous injection of a preparation of partially purified secretin from the extracts of canine antral mucosae resulted in a significant increase in the pancreatic flow in anesthetized rats. We conclude that a small number of secretin cells are, therefore, present in the antral mucosae of dog and rat, and this observation is supported by the presence of an immunologically and biologically active secretin-like molecule with charge and size similar to those of porcine secretin in the canine mucosal extracts.

Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide. Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60.

An Mr 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH2-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).

Vasoactive intestinal peptide receptor activity in human fetal enterocytes.

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind 125I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of 125I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 X 10(-10) M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10(-7) M and above) in the following order of potency: VIP greater than PHI greater than GRF greater than secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9-12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.

Immunochemical studies of the endocrine cells of the gastrointestinal tract. II An immunoperoxide technique for the localization of secretin containing cells in human duodenum.

Endocrine cells containing secretin have been identified in the epithelium lining human duodenum by direct and indirect immunoperoxidase techniques using immune sera raised against pur natural secretin. The techniques were applied to sections of carbodiimide-fixed tissue embedded in polyethylene glycol. Some sections, stained by a modified indirect technique, were processed for electron microscopy; secretin-containing granules were present by ultrastructural preservation was too poor to be of value. The potential advantages of a peroxidase technique over fluorescein-coniugated antisera are discussed.

Catabolism of secretin by the liver and kidney.

We have investigated the roles of the liver and the kidney in the catabolism of secretin, using a specific and sensitive radioimmunoassay. Dogs were prepared with sampling catheters in the aorta, hepatic vein, portal vein, and renal vein and with electromagnetic flow probes on the portal vein, hepatic artery, and renal artery. Secretin levels in the vessels entering and leaving the liver and kidney were determined by radioimmunoassay and the total mass of secretin [concentration (picograms per milliliter) X plasma flow rate (milliliter per minute)] was calculated during an intravenous infusion of exogenous secretin and during release of endogenous secretin by acidification of the proximal intestine. The total masses of secretin entering and leaving the liver were the same during secretin infusion and during the release of endogenous secretin. Under conditions of elevation of plasma secretin, however, the kidney extracted 30 percent of arterial secretin during secretin infusion and 45 percent during release of endogenous secretin. Clearly the kidney is a major site of secretin catabolism.

Secretin in the rat hypothalamo-pituitary system: localization, identification and characterization.

Secretin-like immunoreactivity (SLI) has been identified and characterized in the pituitary of the rat. The concentration in the neurointermediate lobe is about 45 fold higher than the concentration of SLI observed in the anterior lobe. Transections of the pituitary stalk of the rat caused a significant depletion of SLI in the neurointermediate lobe without affecting the content in the anterior lobe. In view of the relatively high concentration of SLI reported to occur in the hypothalamus, it appears that there may be a secretinergic pathway between the brain and the neurointermediate lobe of the pituitary.

Secretin immunoreactivity in rat and pig brain.

Secretin immunoreactivity in rat and pig brain has been identified and characterized utilizing a highly specific radioimmunoassay and fractionation on a high pressure liquid chromatographic system reverse phase column. One immunoreactive peak from each brain extract was observed. Secretin immunoreactivity from rat brain and duodenum coelute, but eluted slightly ahead of the immunoreactivity from pig brain and duodenum and from synthetic porcine secretin. Immunoreactive secretin is widely distributed in the thalamus, hypothalamus and olfactory bulb, cerebral cortex, midbrain, septum, striatum, hippocampus, medulla and pons. The highest concentrations occur in the pineal and the pituitary gland.

The C-terminus ends of secretin and VIP interact with the N-terminal domains of their receptors.

C-terminally truncated secretin and VIP molecules were synthesized, and their ability to occupy the recombinant secretin and VIP1 receptors stably expressed in Chinese hamster ovary (CHO) cells and to stimulate adenylate cyclase activity was studied. On secretin receptors, secretin (1-26) and secretin (1-24) were 10- and 50-fold less potent but as efficient as secretin (1-27); VIP (1-27) was as potent and efficient as VIP (1-28), and VIP (1-26) and VIP (1-25) were both 100-fold less potent. On VIP1 receptor, VIP (1-28) and VIP (1-27) were equipotent and VIP (1-26) and VIP (1-25) were 10- and 300-fold less potent, respectively; secretin (1-27) and secretin (1-26) were of equally low affinity and 10-fold more potent than secretin (1-24). Thus, the secretin and the VIP1 receptors had different selectivity profiles for the recognition of C-terminally truncated secretin and VIP derivatives. The chimeric receptors consisting in the N-terminal part of the secretin receptor on the core of the VIP1 receptor (N-Sn/VIP1.r) and in the N-terminal part of the VIP1 receptor on the core of the secretin receptor (N-VIP1/Sn.r) exhibited the selectivity pattern of the secretin and VIP1 receptors, respectively. The results suggest that the C-terminal end of secretin and VIP interacts with the N-terminal domain of the secretin and VIP receptors.

What may be the anatomical basis that secretin can improve the mental functions in autism?

Autism was first described and characterized as a behavioral disorder more than 50 years ago. The major abnormality in the central nervous system is a cerebellar atrophy. The characteristic histological sign is a striking loss or abnormal development in the Purkinje cell count. Abnormalities were also found in the limbic system, in the parietal and frontal cortex, and in the brain stem. The relation between secretin and autism was observed 3 years ago. Clinical observations by Horváth et al. [J. Assoc. Acad. Minor. Physicians 9 (1998) 9] supposed a defect in the role of secretin and its receptors in autism. The aim of the present work was to study the precise localization of secretin immunoreactivity in the nervous system using an immunohistochemical approach. No secretin immunoreactivity was observed in the forebrain structures. In the brain stem, secretin immunoreactivity was observed in the mesencephalic nucleus of the trigeminal nerve, in the superior olivary nucleus, and in scattered cells of the reticular formation. The most intensive secretin immunoreactivity was observed in the Purkinje cells of the whole cerebellum and in some of the neurons of the central cerebellar nuclei. Secretin immunoreactivity was also observed in a subpopulation of neurons in the primary sensory ganglia. This work is the first immunohistochemical demonstration of secretin-immunoreactive elements in the brain stem and in primary sensory ganglia.

Immunohistochemical evidence of gastro-entero-pancreatic neurohormonal peptides of vertebrate type in the nervous system of the larva of a dipteran insect, the hoverfly, Eristalis aeneus.

Using rabbit and guinea-pig antisera, raised against GEP neurohormonal peptides of mammalian origin, cells were observed in the brain and/or in the fused ventral ganglia of the last (fifth) larval instar of the hoverfly, Eristalis aeneus, being immunoreactive with antisera against insulin, somatostatin, glucagon, PP, secretin, gastrin/CCK/caerulein; substance P, enkephalin and endorphin. Most of these GEP neurohormonal peptides also occurred in nerve fibers. No immunoreactive cells or nerve fibers could be detected with antisera against GIP, VIP, (the central fragments of) CCK, bombesin or neurotensin. The antisera tested failed to reveal any immunoreactive cells or nerves in Weismann's ring (fused corpus allatum/corpus cardiacum and thoracic gland) or in different parts of the alimentary tract. The observations support the hypothesis that neuronal GEP hormonal peptide production in the brain is a genuinely original mechanism and the appearance of endocrine cells in the gut a later feature in evolution.

Presence and possible site of action of secretin in the rat pituitary and hypothalamus.

Secretin-like immunoreactivity was detected in extracts of several rat brain structures by radioimmunoassay, most notably in the pituitary, hypothalamus, pineal and septum. Its localization to these structures suggested that it might play a role in neuroendocrine events similar to its structural homolog vasoactive intestinal peptide. Dose-related stimulations (MED, 10(-7) M) of prolactin (PRL) release were observed after incubation of synthetic secretin with dispersed, cultured pituitary cells from male and ovariectomized (OVX) female rats. In OVX females, i.v. infusion of a high dose of secretin (10 micrograms) resulted in a significant elevation of PRL levels. Doses of secretin as low as 0.1 micrograms when administered into the third cerebroventricle were capable of significantly inhibiting PRL release in both males and OVX females, suggesting an ultrashort-loop, negative feedback of secretin. Secretin can now be added to the growing list of putative PRL-releasing agents.

Inhibition of gastric acid secretion by intravenous cholecystokinin extract.

The rapid single intravenous injection of the cholecystokinin preparation Cecekin (Vitrum) was found to inhibit the responses of Heidenhain pouch dogs to stimulation by gastrin extract or synthetic gastrin-like penta-peptide at both submaximal and supramaximal dose rates, to histamine, to the stable cholinergic drug Amechol, and to feeding a meat meal. The pattern of inhibition suggested that the inhibitor effect was exerted in the region of the acid secreting cell. Pure cholecystokinin was capable of inhibiting the acid response of the Heidenhain pouch dogs to histamine. Although both pure secretin and pure cholecystokinin have now been demonstrated to be possible inhibitor agents arising from duodenal mucosa, it is likely that endogenous duodenal acidification releases a further as yet unidentified humoral inhibitor agent, or that secretin and/or cholecystokinin may act in collaboration with each other or a further inhibitor agent. In dogs with both a Heidenhain pouch and a simple fistula into the normally vagally innervated gastric remnant and in which antrectomy had been performed, a single rapid intravenous injection of the Cecekin had no effect on the Heidenhain pouch responses to the continuous intravenous infusion of histamine. The acid output from the main gastric remnant, however, was increased by the Cecekin injection. Several hypotheses to account for the difference in behaviour in the two preparations are discussed. The possibility is raised that Cecekin contains some gastrin-like activity.

Abnormalities of small intestinal endocrine cells in non-obese diabetic mice.

The endocrine cells in the duodenum of pre-diabetic and diabetic female non-obese diabetic (NOD) mice aged 22-24 weeks were studied by means of immunohistochemistry and computed image analysis as well as by radioimmunoassays of tissue extracts. As controls, 12 female BALB/cJ mice of the same age as NOD mice were used. The number of secretin-immunoreactive cells increased in diabetic but not in pre-diabetic NOD mice. The level of extractable secretin was higher in both pre-diabetic and diabetic NOD mice. The number of GIP-, CCK/gastrin-, and serotonin-immunoreactive cells was significantly reduced in both pre-diabetic and diabetic NOD mice. There was no statistical difference in the number of somatostatin-immunoreactive cells between the NOD mice and controls. The level of GIP was higher and gastrin was lower in NOD mice compared to controls. There was no statistical difference in the somatostatin level between the NOD mice and controls. The cell secretory index was elevated in all the endocrine cell types except CCK/gastrin cells. It has been suggested that some of the changes in the duodenal endocrine cells could be attributed to the diabetes state, but most of the changes seem to take place before the onset of diabetes. The abnormalities in the duodenal endocrine cells observed here in an animal model for diabetes type I might have relevance for the gastrointestinal dysfunction displayed in human diabetes.

Islet cell tumors of the pancreas and the alimentary tract.

Functioning tumors of the pancreatic islets are now recognized as the source of clinical syndromes affecting the gastrointestinal tract which have a wide variety of catastrophic symptoms. Experiences with thirty-six cases suggest at least four separate diagnostic categories in the ulcerogenic tumor syndrome. These include: a typical history, gastric analysis, and roentgenographic findings with boderline fasting serum gastrin levels; ulcerogenic tumor with evidence of hyperparathyroidism; iatrogenic ulcerogenic syndrome associated with failure of a previous operation for duodenal ulcer; and the classic ulcerogenic syndrome associated with a fulminating ulcer diathesis or diarrhea and high serum gastrin levels. The problems presented at operation include: decisions to be make in the presence of a negative exploration; the finding of a solitary tumor in the wall of the duodenum; solitary pancreatic tumors particularly in the body and tail; ulcerogenic tumors in the very young; liver metastases in the elderly; and the wisdom of removing gross metastases in combination with total gastrectomy. The long-term survival in the ulcerogenic tumor syndrome approximated 50 per cent, with 40 per cent of those having proved malignancy living five years. Evidence of hyperparathyroidism is relatively common in association with both the ulcerogenic and the diarrheogenic tumor syndromes. The association may by a result of a congenital abnormality, metabolic alkalosis, or a direct effect of the islet cell tumor. Parathyroidectomy may be indicated when both the serum calcium and parathormone levels are elevated in the presence of borderline fasting gastrin levels. The latter may return to normal after parathyroidectomy. The evidence of hyperparathyroidism closely parallels the episodes of diarrhea in the diarrheogenic syndrome, and hyperparathyroidism may regress spontaneously after total removal of the pancreatic tumor. Just as routine calcium determinations made the diagnosis of hyperparathyroidism more commonplace, it is suggested that the gastrointestinal syndromes associated with islet cell tumor would receive wider recognition if radioimmunoassays for gastrin as well as secretin, and the other secretin-like polypeptides, were carried out routinely.