Involvement of calmodulin and protein kinase C in cholecystokinin release by bombesin from STC-1 cells

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.     

Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine.

Pancreatic proteases in the duodenum inhibit the release of cholecystokinin (CCK) and thus exert feedback control of pancreatic exocrine secretion. Exclusion of proteases from the duodenum either by the diversion of bile-pancreatic juice or by the addition of protease inhibitors stimulates exocrine pancreatic secretion. The mechanism by which pancreatic proteases in the duodenum regulate CCK secretion is unknown. In this study, we isolated a trypsin-sensitive peptide that is secreted intraduodenally, releases CCK, and stimulates pancreatic enzyme secretion in rats. This peptide was found to be identical to the porcine diazepam binding inhibitor by peptide sequencing and mass spectrometry analysis. Intraduodenal infusion of 200 ng of synthetic porcine diazepam binding inhibitor1-86 in rats significantly stimulated pancreatic amylase output. Infusion of the CCK antagonist MK-329 completely blocked the diazepam binding inhibitor-stimulated amylase secretion. Similarly, diazepam binding inhibitor33-52 [corrected] also stimulated CCK release and pancreatic secretion in a dose-dependent manner although it was 100 times less potent than the whole peptide. Using a perfusion system containing isolated mucosal cells from the proximal intestine of rats, porcine diazepam binding inhibitor 10(-12) M) dose dependently stimulated CCK secretion. In separate studies, it was demonstrated that luminal secretion of the diazepam binding inhibitor immunoreactivity (7.5 X 10(11) M) could be detected in rat's intestinal washing following the diversion of bile-pancreatic juice. The secretion of this peptide was inhibited by atropine. In conclusion, we have isolated and characterized a CCK-releasing peptide that has a sequence identical to the porcine diazepam binding inhibitor from pig intestinal mucosa and that stimulates CCK release when administered intraduodenally in rat. This peptide may mediate feedback regulation of pancreatic enzyme secretion.     

Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion.

Cholecystokinin (CCK) secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition of CCK release caused by pancreatic proteases is due to proteolytic inactivation of a CCK-releasing peptide present in intestinal secretion. To purify the putative luminal CCK-releasing factor (LCRF), intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in conscious rats. From these secretions, the peptide was concentrated by ultrafiltration followed by low-pressure reverse-phase chromatography and purified by reverse-phase high-pressure liquid chromatography. Purity was confirmed by high-performance capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially purified fractions strongly stimulated both pancreatic secretion and CCK release while CCK receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for 41 residues as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV. When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in conscious rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRF-(1-6) abolished the CCK releasing activity of intestinal secretions. These studies demonstrate, to our knowledge, the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.     

CCK-releasing activity of rat intestinal secretion: effect of atropine and comparison with monitor peptide

A bioassay for studying the cholecystokinin (CCK)-releasing activity of intraluminal protease-sensitive bioactive peptides was developed. In conscious rats, bile and pancreatic juice were chronically diverted from the proximal intestine to the ileum to cause chronic stimulation of CCK release and pancreatic protein secretion. CCK-releasing activity of test substances was assayed during transient inhibition of CCK release by intraduodenal sodium taurocholate (78 mumols/h). Intestinal secretion as a source of the putative trypsin-sensitive intestinal CCK-releasing peptide was obtained by rapid intestinal perfusion of isolated Thiry-Vella fistulae of jejunum in conscious rats, collected with or without atropine pretreatment. Partially purified rat pancreatic secretory trypsin inhibitor (PSTI, or "monitor peptide") was compared with ovomucoid trypsin inhibitor (OMTI) and with concentrated jejunal secretions for CCK-releasing activity and trypsin inhibitor activity. Concentrated, heat-treated jejunal secretions were the strongest stimulants of CCK release and pancreatic protein secretion in this model. OMTI had no CCK-releasing activity in this model, whereas a larger amount (approximately 5x, based on trypsin inhibitor activity) of PSTI weakly but significantly stimulated CCK release. CCK-releasing activity manifested by pancreatic protein secretion was equivalent in intestinal washes from atropine-treated and control Thiry-Vella fistula donor rats. Concentrated jejunal secretions had no trypsin inhibitory activity, indicating that the putative intestinal CCK-releasing peptide and "monitor peptide" are different substances.     

Isolation and bioactivity of putative cholecystokinin-releasing peptide from rat small intestinal mucosa

Pancreatic exocrine secretion in the conscious rat is regulated by proteases in the intestine secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in the mechanism. The authors proposed that the release of CCK was regulated by a CCK-releasing factor secreted into the intestinal lumen from the proximal intestine. We isolated and partially purified a CCK-releasing factor from rat small intestine by gel filtration and high performance liquid chromatography. The partially purified CCK-releasing factor increased pancreatic exocrine secretions and plasma CCK concentrations in conscious rats and this activity was abolished after the incubation with trypsin. The bioactivity of the partially purified CCK-releasing factor was confirmed.     

Isolation and bioactivity of putative cholecystokinin-releasing peptide from rat small intestinal mucosa.

Pancreatic exocrine secretion in the conscious rat is regulated by proteases in the intestine secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in the mechanism. The authors proposed that the release of CCK was regulated by a CCK-releasing factor secreted into the intestinal lumen from the proximal intestine. We isolated and partially purified a CCK-releasing factor from rat small intestine by gel filtration and high performance liquid chromatography. The partially purified CCK-releasing factor increased pancreatic exocrine secretions and plasma CCK concentrations in conscious rats and this activity was abolished after the incubation with trypsin. The bioactivity of the partially purified CCK-releasing factor was confirmed.     

Control of glucagon-like immunoreactive peptide secretion from fetal rat intestinal cultures

Some of the mechanisms underlying intestinal glucagon-like immunoreactive (GLI) peptide secretion from cultured fetal rat intestinal cells were investigated using modulators of the adenylate cyclase pathway [(Bu)2cAMP, theophylline, isobutylmethylxanthine], calcium fluxes (ionomycin, A23187), and protein kinase-C (phorbol ester). All of these agents were found to stimulate GLI peptide release, to 120-230% of paired control values (P less than 0.05-0.001). (Bu)2cAMP, but not the phorbol ester, also increased the total cell content of GLI peptides over the 2-h incubation period (P less than 0.05). No synergism between any of the three pathways was detected. When the mol wt distribution of the stored and secreted GLI peptides was determined in control and (Bu)2 cAMP-stimulated samples, 68 +/- 2% of the peptide corresponded to glicentin, while the remainder eluted with the same distribution coefficient as oxyntomodulin. No 3.5K glucagon was detected in any of the extracts. GLI peptide secretion by the cells was not altered by several pancreatic glucagon secretagogues (cortisol, bombesin, and prostaglandins E1 and D2), but was stimulated by the opioid peptide beta-endorphin (1 microM; P less than 0.02). These studies have indicated that the control of secretion of fetal rat intestinal GLI peptides is complex, involving activation of any one or a combination of the three major second messenger systems. A role for the adenylate cyclase pathway in regulating GLI peptide biosynthesis is also suggested.     

Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phosphate production

Calcitonin is present in both the hypothalamus and pituitary of the rat, and normal rat anterior pituitary cells express calcitonin receptors. Calcitonin has been reported to inhibit or to stimulate PRL release from rat anterior pituitary cells. We have investigated the effects of salmon calcitonin on basal and stimulated PRL release from rat anterior pituitary cells and have studied the effects of this peptide on the intracellular biochemical pathways involved in PRL release. Salmon calcitonin had no significant effect on basal PRL release, but inhibited (P less than 0.01) TRH-stimulated PRL release without affecting PRL release promoted by angiotensin II, neurotensin, phorbol myristate acetate (a protein kinase C activator), or maitotoxin (a calcium channel activator). Salmon calcitonin had no effect on the increase in PRL release and intracellular cAMP concentration after exposure of pituitary cells to vasoactive intestinal peptide or forskolin. Salmon calcitonin significantly decreased (P less than 0.01) the TRH-stimulated rise in inositol phosphates without affecting the angiotensin II-stimulated increase in inositol phosphates. Similarly, salmon calcitonin decreased the TRH-stimulated increase in cytosolic calcium and arachidonate liberation by pituitary cells. We conclude that salmon calcitonin selectively decreases TRH-stimulated PRL release by a mechanism that involves a decrease in inositol phosphate production, as well as a subsequent reduction in cytosolic calcium levels and in arachidonate liberation.     

In vitro secretion of immunoreactive tonin from dispersed rat submandibular gland cells

Dispersed cells from the submandibular gland of the male rat were prepared by collagenase treatment to study the mechanism by which immunoreactive tonin is secreted in vitro. Norepinephrine, epinephrine, and phenylephrine stimulated tonin release, an effect that was inhibited by phentolamine but not by propranolol, whereas isoproterenol, carbachol, histamine, and serotonin did not stimulate tonin release. The stimulatory effect elicited by alpha-adrenergic agonists was inhibited by both removal of Ca2+ from the medium and addition of diltiazem and nifedipine, both selective calcium channel blockers. The divalent cation ionophore A23187 stimulated tonin release in the presence of Ca2+, but not in the presence of Mg2+. Dibutyryl cyclic adenosine 3',5'-monophosphate, methylisobutylxanthine, angiotensin II, and vasoactive intestinal peptide had no effect on tonin release. The apparent molecular size of immunoreactive tonin released into the medium under basal and norepinephrine-stimulated conditions was similar to that of standard tonin by gel exclusion chromatography. These data suggest that the in vitro secretion of immunoreactive tonin from rat submandibular gland is initiated by activation of alpha-adrenergic receptors and apparently involves a mechanism dependent not on cyclic adenosine 3',5'-monophosphate, but on the influx of extracellular Ca2+.     

Expression of cholecystokinin A receptors in neurons innervating the rat stomach and intestine.

BACKGROUND & AIMS: Two distinct receptors, cholecystokinin (CCK)-A and CCK-B, mediate CCK effects in the digestive system. The aim of this study was to elucidate the cellular sites of expression of CCK-A receptor in the rat stomach and small intestine. METHODS: We developed and characterized an antibody to the N-terminal region (LDQPQPSKEWQSA) of rat CCK-A receptor and used it for localization studies with immunohistochemistry. RESULTS: Specificity of the antiserum was demonstrated by (1) detection of a broad band at 85-95 kilodaltons in Western blots of membranes from CCK-A receptor CHO-transfected cells; (2) cell surface staining of CCK-A receptor-transfected cells, (3) translocation of CCK-A receptor immunostaining in CCK-A receptor-transfected cells after exposure to CCK; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with the peptide used for immunization. CCK-A receptor immunoreactivity was localized to myenteric neurons and to fibers in the muscle and mucosa. In the stomach, myenteric neurons and mucosal fibers were abundant. Many CCK-A receptor myenteric neurons contained the inhibitory transmitter vasoactive intestinal polypeptide, and some were immunoreactive for the excitatory transmitter substance P. Subdiaphragmatic vagotomy reduced the density of CCK-A receptor fibers in the gastric mucosa by approximately 50%, whereas celiac/superior mesenteric ganglionectomy had no detectable effect on fiber density. CONCLUSIONS: CCK-A receptor is expressed in functionally distinct neurons of the gastrointestinal tract. CCK-A receptor may mediate reflexes stimulated by CCK through the release of other transmitters from neurons bearing the receptor.     

Effects of verapamil on amylase release from rat pancreatic acini and its relation to calcium fluxes

The effects of verapamil on amylase release and Ca2+ fluxes from rat pancreatic acini have been studied. Verapamil at concentrations above 10 microM dose-dependently inhibited amylase release stimulated by carbachol, but enhanced the amylase release stimulated by cholecystokinin (CCK) and secretin. Verapamil had no significant effect on calcium uptake induced by carbachol or CCK, but significantly inhibited Ca+2 efflux caused by carbachol and slightly increased that caused by CCK. In a Ca2+-free, EDTA-containing medium, the increase in cytoplasmic free Ca2+ caused by carbachol was significantly inhibited by verapamil. Verapamil alone up to 400 microM had no effect on the release of lactic dehydrogenase. In conclusion, the effect of verapamil on amylase release from rat pancreatic acini differs depending on the type of secretagogue used to stimulate amylase release. This effect is not related to blockage of Ca2+ uptake, indicating another mechanism of verapamil on pancreatic acini.     

Regulation of intestinal mast cells and luminal protein release by cerebral thyrotropin-releasing hormone in rats

BACKGROUND & AIMS: Intestinal mast cell activity is modulated by the central nervous system, but the mechanisms are not well established. The aim of this study was to investigate whether cerebral thyrotropin- releasing hormone (TRH) activates intestinal mast cells and to elucidate the mechanisms involved, specifically, the contribution of mast cells to vagally stimulated luminal protein release. METHODS: In anesthetized rats, mast cell activation was assessed by measuring the release of the specific mucosal rat mast cell protease II (RMCP II) and prostaglandin (PG) D2 into the intestinal lumen. Luminal protein release was measured as an index of epithelial permeability to macromolecules. RESULTS: Intracisternal injection of the TRH analogue RX 77368 (30 ng) induced a transient increase in intestinal release of RMCP II and PGD2 that was abolished by dox-antrazole. RX 77368- stimulated RMCP II release was also abolished by vagotomy and reduced by atropine by 65%. However, both systemic capsaicin and indo-methacin enhanced RMCP II release. RX 77368-stimulated luminal protein release was abolished by vagotomy and reduced by doxantrazole. CONCLUSIONS: Central vagal activation by TRH stimulates intestinal mast cell secretion, in part via peripheral muscarinic receptors, and is modulated by PGs and capsaicin-sensitive afferent innervation. Intestinal mast cell activation contributes to the TRH analogue- stimulated luminal protein release. (Gastroenterology 1996 Dec;111(6):1465-73)     

Effect of pancreastatin on pancreatic endocrine and exocrine secretion

Pancreastatin is a novel peptide that was recently purified from extracts of the porcine pancreas. The present study shows that pancreastatin (10(-9)-10(-8) M) can stimulate release of insulin from both the isolated perfused rat pancreas and from cultured rat islet cells in the presence of a low, non-insulinotropic concentration of glucose (4.2 mM). Pancreastatin (10(-9) M) can also inhibit release of insulin stimulated by a high concentration of glucose (16.7 mM). Pancreastatin, at 10(-8) M, can enhance glucose (8.3 mM) induced release of insulin in the static islet cell incubation. In addition, pancreastatin (10(-9)-10(-8) M) can inhibit, in a dose-dependent fashion, cholecystokinin (CCK)-8 stimulated release of amylase from dispersed guinea pig pancreatic acini. Pancreastatin alone, however, did not affect basal release of amylase. Our study shows that pancreastatin can exert a direct effect on both pancreatic endocrine and exocrine secretion.     

Regulation of intestinal concentration of cholecystokinin by bile and/or pancreatic juice

Pancreatic exocrine secretion in conscious rats is regulated by intraluminal bile and/or pancreatic juice. Exclusion of bile and/or pancreatic juice from the intestinal lumen caused cholecystokinin (CCK) release and stimulated pancreatic secretion. CCK in the plasma is mainly derived from endocrine cells in the proximal small intestinal mucosa. We examined the changes in CCK concentrations in the intestinal mucosa and compared them to those of plasma CCK concentrations and the changes of luminal trypsin activities after bile and/or pancreatic juice diversion in conscious rats. Rats with bile and pancreatic fistulae were used. Each treatment of bile, pancreatic juice, and bile-pancreatic juice diversion decreased luminal trypsin activity and increased plasma and intestinal CCK concentrations. The potency of the stimulatory effect on plasma and intestinal CCK concentrations was bilepancreatic juice diversion>pancreatic juice diversionbile diversion. Neither plasma CCK concentration nor intestinal CCK concentration was in inverse proportion to trypsin activity. The plasma CCK concentration did not parallel intestinal CCK concentration. Intravenous infusion of CCK-8 (300 pmol/kg/hr) did not increase CCK concentration in the intestinal mucosa. It was proposed that bile and/or pancreatic juice in the intestinal lumen regulated CCK concentrations not only in the plasma but also in the intestinal mucosa.     

Vasoactive intestinal polypeptide stimulates parathyroid hormone release by interaction with cyclic adenosine monophosphate production of bovine parathyroid cells.

Influence of vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, and substance P was investigated on dispersed parathyroid cells of adult cattle. At a physiological concentration of extracellular calcium, vasoactive intestinal polypeptide stimulated the parathyroid hormone release in a dose-dependent manner, whereas no effects were noted for the other peptides. The dependency of PTH secretion upon extracellular calcium was shifted to the right by vasoactive intestinal polypeptide at 10(-6) mol/l, with a tendency for greater effects at low (0.5 mmol/l) than high concentrations (2.0-3.0 mmol/l) of the cation. Vasoactive intestinal polypeptide significantly enhanced cAMP release of the parathyroid cells, whereas no influence was noted on cytoplasmic calcium or pH within the cells. The results suggest that vasoactive intestinal polypeptide stimulates the PTH release by interaction with cAMP production of the parathyroid cells. This effect may contribute to the development of hypercalcemia in patients with neuroendocrine tumours secreting vasoactive intestinal polypeptide.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

Apelin, a new enteric peptide: localization in the gastrointestinal tract, ontogeny, and stimulation of gastric cell proliferation and of cholecystokinin secretion

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. The aim of this study was to characterize apelin expression (mRNA levels) in the rat gastrointestinal tract and pancreas, to localize distribution of apelin peptide-containing cells in the stomach by immunohistochemistry, and to characterize the ontogeny of gastric apelin expression and peptide and the influence of apelin on gastric cell proliferation in vitro. Additionally, the effect of apelin on cholecystokinin (CCK) secretion and the involvement of MAPK, protein kinase C, and changes in intracellular Ca(2+) in apelin-induced CCK secretion in vitro were examined. Northern analysis showed a maximal apelin expression in the stomach with a lower expression level in the intestine. Apelin expression was not detected in the pancreas. Immunohistochemistry revealed abundant apelin-positive cells in the glandular epithelium of the stomach. The ontogeny study showed a higher apelin expression in the fetal and postnatal rat stomachs when compared with the adult stomach. In contrast to apelin expression, apelin peptide was not detected in the rat stomach until 20 d of age and then increased progressively with age. Apelin was shown to stimulate gastric cell proliferation in vitro. Apelin also stimulated CCK secretion from a murine enteroendocrine cell line (STC-1); apelin-stimulated CCK secretion is mediated through MAPK but not by intracellular Ca(2+) signaling. Together, these data indicate that apelin is an important new stomach peptide with a potential physiological role in the gastrointestinal tract.     

Starvation, leptin and epithelial cell proliferation in the gastrointestinal tract of the mouse

BACKGROUND/AIMS: Leptin, the ob/ob gene product, is a recently discovered peptide hormone, secreted by adipocytes, which can act as a satiety factor to regulate food intake. Its levels thus will be related to the presence of food in the lumen of the gut, and food intake is one of the most potent stimuli for intestinal epithelial cell proliferation. Leptin has a variety of other actions and the aim of this study was to see if one of these was to stimulate mucosal growth. METHODS: Three groups of mice were fed ad libitum, starved for 48 h or starved for 48 h and given twice-daily intraperitoneal injections of recombinant leptin (1 microg/g). RESULTS: Starvation led to a 20% decrease in body weight and a similar decrease in the weights of the intestines. Starvation also markedly inhibited intestinal epithelial cell proliferation. Leptin had little effect on the small intestine and did not stimulate proliferation. However, in the hind gut it was associated with small but significant decreases in caecal weight, distal colon mitotic counts (p = 0.036) and in colonic crypt area (approximately 20%, p<0.001). CONCLUSION: Leptin did not stimulate intestinal cell proliferation, however it did have a paradoxical inhibitory action on the caecum and colon.