cholecystokinin and Intestinal-Neoplasms

Neuroendocrine tumors in the pancreas and the gastrointestinal tract may secrete hormones which cause specific syndromes. Well-known examples are gastrinomas, glucagonomas, and insulinomas. Cholecystokinin-producing tumors (CCKomas) have been induced experimentally in rats, but a CCKoma syndrome in man has remained unknown until now.. Using a panel of immunoassays for CCK peptides and proCCK as well as for chromogranin A, we have examined plasma samples from 284 fasting patients with gastroenteropancreatic neuroendocrine tumors. In hyperCCKemic samples, plasma CCK was further characterized by chromatography.. One of the patients displayed gross hyperCCKemia. She was a 58-year old woman with a pancreatic endocrine tumor, liver metastases, 500-1000-fold elevated basal CCK concentration in plasma, diarrhea, severe weight loss, recurrent peptic ulcer and bilestone attacks from a contracted gallbladder. The CCK concentrations in plasma were not affected by resection of the pancreatic tumor, but decreased to normal after hemihepatectomy with removal of the metastases.. A CCKoma syndrome with severe hypersecretion of CCK exists in man. The duodenal ulcer disease and diarrhea with permanently low gastrin in plasma suggest that CCKomas may mimic gastrinoma-like symptoms, because CCK peptides are full agonists of the gastrin/CCK-B receptor.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Child; Cholecystokinin; Denmark; Female; Gastrinoma; Gastrins; Humans; Intestinal Neoplasms; Liver Neoplasms; Male; Middle Aged; Neuroendocrine Tumors; Pancreas; Pancreatic Neoplasms; Rats; Stomach Neoplasms; Young Adult

The mouse intestinal neuroendocrine tumor cell line STC-1 secretes cholecystokinin (CCK) and other hormones. We investigated the role of Ca2+, calmodulin (CaM), and protein kinase C (PKC) in the regulation of CCK release from STC-1 cells. Phorbol 12-myristate 13-acetate (TPA) significantly stimulated CCK release. Staurosporine significantly inhibited CCK release from STC-1 cells stimulated by TPA in a dose-dependent manner. The absence of extracellular calcium completely inhibited CCK release from TPA-stimulated STC-1 cells. Neurotensin did not stimulate CCK release from these cells. W-7, a CaM antagonist, reduced CCK release from STC-1 cells stimulated by bombesin in a dose-dependent manner. These findings suggest that CaM and PKC play an important role in the regulation of CCK release from STC-1 cells stimulated by bombesin.

Topics: Animals; Bombesin; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cholecystokinin; Enzyme Activation; Enzyme Inhibitors; Intestinal Neoplasms; Mice; Neuroendocrine Tumors; Neurotensin; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Although GABA(C) receptors play a crucial role in the mammalian central nervous system, their functional expression in peripheral tissues has not yet been studied. Using the gut neuroendocrine tumor cell line STC-1 as a model, we provide first evidence for the functional expression of GABA(C) receptors in the gut: mRNAs of the GABA(C) receptor subunits rho1 and rho2 were detected in STC-1 cells by reverse transcription polymerase chain reaction (RT-PCR). Applying anti-rho-antibodies, specific immunostaining for GABA(C) receptors was observed. For functional characterization, the effects of GABA(C) receptor activation on [Ca2+]i and hormone secretion were studied. The selective GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA) induced dose-dependent increases both of [Ca2+]i and of hormone (cholecystokinin) secretion. The stimulatory effects of CACA were antagonized by the GABA(C) receptor blockers (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and 3-aminopropyl(methyl)phosphinic acid (3-APMPA). These results demonstrate that GABA(C) receptors play an important role in neuroendocrine gastrointestinal secretion.

Topics: Acrylates; Animals; Bicuculline; Calcium; Cholecystokinin; Cytarabine; GABA Antagonists; gamma-Aminobutyric Acid; Gene Expression; Immunohistochemistry; Intestinal Neoplasms; Kinetics; Mice; Mice, Transgenic; Neuroendocrine Tumors; Organophosphorus Compounds; Pyridazines; Receptors, GABA; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.

Topics: Amiloride; Animals; Antigens, Polyomavirus Transforming; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cell Membrane; Cell Membrane Permeability; Cholecystokinin; Diltiazem; Hydrogen-Ion Concentration; Insulin; Intestinal Neoplasms; Mice; Mice, Transgenic; Polyomavirus; Potassium; Potassium Channels; Promoter Regions, Genetic; Rats; Simian virus 40; Sodium; Sodium-Hydrogen Exchangers

Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.

Topics: Animals; Aspartic Acid Endopeptidases; Cholecystokinin; Chromatography, Gel; Insulinoma; Intestinal Neoplasms; Models, Chemical; Oligonucleotides, Antisense; Pancreatic Neoplasms; Peptide Fragments; Proprotein Convertases; Rats; Sincalide; Tumor Cells, Cultured

Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing. Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.

Topics: Cholecystokinin; Gene Expression; Intestinal Neoplasms; Pancreatic Neoplasms; Peptide Fragments; Proprotein Convertase 2; RNA, Antisense; RNA, Messenger; Sincalide; Subtilisins; Transfection; Tumor Cells, Cultured

Secretion of cholecystokinin (CCK) from the endocrine cells of small intestinal mucosa and the murine intestinal tumor cell line STC-1 is known to involve both adenosine 3',5'-cyclic monophosphate (cAMP)-and Ca(2+)-dependent signal transduction pathways. However, the endogenous stimulant(s) that acts through the cAMP-dependent cascade has not been identified. We determined the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on CCK secretion and cAMP production and its interaction with other CCK secretagogues in STC-1 cells. At concentrations > 10 nM, PACAP-27 stimulated the release of large intestinal CCK from STC-1 cells in a time- and dose-dependent manner. The stimulatory effect of PACAP-27 was enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). PACAP-27, PACAP-38, and vasoactive intestinal polypeptide (VIP), with or without IBMX, were equally effective and potent to elicit CCK release with similar half-maximal doses and maximal levels of stimulation. Both forms of PACAP and VIP stimulated a transient but not significant increase in the cellular cAMP level. In the presence of IBMX, all three peptides increased significantly the cellular cAMP level between 2 and 5 min, but PACAP produced a two times higher level than VIP. The stimulatory effect of PACAP-27 on CCK release was also potentiated by bombesin and KCl but without a synergistic production of cAMP. With or without IBMX, PACAP-27-stimulated CCK secretion was not affected by the Ca2+ channel blocker diltiazem (1 microM), the cell-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 25 microM), or by downregulation of protein kinase C. The stimulatory effects of KCl and bombesin were either reduced or abolished by these treatments. The synergistic effect of bombesin with PACAP was abolished by diltiazem and BAPTA-AM but not by downregulation of protein kinase C, whereas KCl remained synergistic with PACAP after these treatments. Taken together, these results indicate that PACAP may be a neuromodulator of CCK secretion that acts through activation of adenylate cyclase and may function as a coregulator with other CCK secretagogues that are known to increase intracellular Ca2+ concentration.

Topics: Animals; Cholecystokinin; Cyclic AMP; Intestinal Mucosa; Intestinal Neoplasms; Intestine, Small; Mice; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Signal Transduction; Tumor Cells, Cultured

A procedure for processing-independent quantitation of procholecystokinin (proCCK) and its products has been applied to plasma. The procedure is based on tryptic cleavage after Lys61 and Arg71 with subsequent monospecific radioimmuno-analysis of fragment 62-71 of human proCCK, which again corresponds to fragment 1-10 of CCK-22. The detection limit of the analysis was 0.2 pmol/l. Plasma was extracted with ethanol. In plasma from 13 healthy volunteers the basal concentration with the above-mentioned radioimmunoassay was 1.1 +/- 0.1 pmol/l (mean +/- S.E.M.) before, and 13.7 +/- 0.6 pmol/l after, incubation with trypsin. Two hours after ingestion of a mixed meal, the plasma concentration was 2.0 +/- 0.1 pmol/l before, and 21.7 +/- 1.2 pmol/l after tryptic cleavage. With a conventional CCK radioimmunoassay specific for the C-terminally amidated and O-sulfated bioactive epitope, the concentration was 1.0 +/- 0.1 pmol/l in the basal state and 4.2 +/- 0.4 pmol/l 2 h after a meal. Tryptic cleavage did not increase the concentrations of amidated, bioactive CCK peptides. In plasma from 37 patients with the carcinoid syndrome, the basal concentration of proCCK and its products was 14.1 (2.8-150.4) pmol/l (median (range)), compared with 0.3 (0-18.8) pmol/l for carboxyamidated CCK. Only two patients had significantly elevated CCK concentrations. We conclude that processing-independent analysis is useful for quantitation of proCCK and its products in plasma, since it quantitates CCK cell secretion more accurately than conventional CCK assays.

Topics: Adult; Cholecystokinin; Chromatography, Gel; Duodenum; Female; Humans; Hydrolysis; Intestinal Mucosa; Intestinal Neoplasms; Male; Malignant Carcinoid Syndrome; Middle Aged; Peptides; Plasma; Protein Precursors; Protein Processing, Post-Translational; Radioimmunoassay; Trypsin

Pancreas and gut hormones are involved in many endocrine and gastrointestinal diseases. Radioimmunoassays for these hormones have proved particularly valuable in diagnosis, localisation and control of treatment of endocrine tumours, of which many are mixed. An estimate based on ten years experience in a homogenous population of 5 million inhabitants (Denmark) suggests, that endocrine gut tumour-syndromes on an average appear with an incidence of 1 patient per year/syndrome/million. At present six different syndromes are known: 1) The insulinoma syndrome, 2) The Zollinger-Ellison syndrome.3) The Verner-Morrison syndrome. 4) The glucagonoma syndrome. 5) The somatostatinoma syndrome, and 6) the carcinoid syndrome. Accordingly diagnostically valuable RIAs for pancreas and gut hormones include those for insulin, gastrin, VIP, HPP, glucagon, somatostatin, and presumably also substance P. It is probably safe to predict that the need for gut and pancreas hormone RIAs within the next decade will increase greatly in order to assure proper management of tumours producing gastroentero-pancreatic hormones.

Topics: Adenoma, Islet Cell; Carcinoid Tumor; Cholecystokinin; Gastric Inhibitory Polypeptide; Gastrins; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptides; Humans; Insulin; Intestinal Neoplasms; Motilin; Pancreatic Hormones; Pancreatic Neoplasms; Pancreatic Polypeptide; Radioimmunoassay; Secretin; Somatostatin; Substance P; Vasoactive Intestinal Peptide; Zollinger-Ellison Syndrome

Topics: Animals; Carcinoid Tumor; Cholecystokinin; Dogs; Enterochromaffin Cells; Gastric Inhibitory Polypeptide; Gastric Mucosa; Gastrins; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptides; Humans; Intestinal Mucosa; Intestinal Neoplasms; Pancreatic Polypeptide; Rats; Secretin; Somatostatin; Stomach Neoplasms; Vasoactive Intestinal Peptide