Cyclic AMP-dependent protein kinase A and EPAC mediate VIP and secretin stimulation of PAK4 and activation of Na+,K+-ATPase in pancreatic acinar cells.

Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na+,K+-ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na+,K+-ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na+,K+ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.

Regulation of the human secretin gene is controlled by the combined effects of CpG methylation, Sp1/Sp3 ratio, and the E-box element.

To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (-11 to -341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (-336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5'-Aza-2' deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

Pancreatic secretory response to feeding in the calf: CCK-A receptors, but not CCK-B/gastrin receptors are involved.

In bovine species, as in human, the pancreas predominantly expresses cholecystokinin-B (CCK-B)/gastrin receptors. However, the role of this receptor in the regulation of meal-stimulated pancreatic enzyme release has not been determined. In milk-fed calves, we previously described prandial patterns of exocrine pancreatic secretion and a long prefeeding phase was observed. The present study was aimed at determining both the role of external stimuli in the outset of the prefeeding phase and the implication of pancreatic CCK-A and CCK-B/gastrin receptors in the mediation of pancreatic response to feeding. The first objective was studied by suppressing external stimuli associated with food intake (unexpected meal) and the second by infusing highly specific and potent antagonists of CCK-A (SR 27897) and CCK-B/gastrin (PD 135158) receptors during the prandial period. When calves were given an unexpected meal, the long prefeeding increase in pancreatic secretion was absent. SR 27897 (but not PD 135158) inhibited the preprandial phase and greatly reduced postprandial pancreatic juice and enzyme outflows. The expectancy of a meal seemed to elicit an increased pancreatic response right before a meal and CCK-A receptors may mediate this information via neural pathways. The implication of CCK and CCK-A receptors in mediating the postfeeding pancreatic response was also demonstrated. The participation of CCK-B/gastrin receptors in this regulation was not demonstrated.

Suppression of histidine decarboxylase activity in rat oxyntic mucosa by beraprost sodium, a prostacyclin analogue.

Prostaglandins (PGs) affect various aspects of gastric functions. In the present study the orally administered PGI2 derivative beraprost sodium (TRK-100.1 micrograms per kg body weight) decreased oxyntic histidine decarboxylase activity without changing serum gastrin levels. Antral pH increased 4 hr after treatment. Beraprost also decreased the pentagastrin-induced histidine decarboxylase activity at the same dose. Serum levels of secretin, somatostatin and glucose, and oxyntic mucosal levels of histamine and somatostatin, showed no significant change after treatment with beraprost. These results suggest that the response of oxyntic histidine decarboxylase to gastrin is modified by one or more prostanoids including PGI2. This mechanism might play a role in gastric mucosal protection.

Importance of gastrin-releasing peptide on acid-induced secretin release and pancreaticobiliary and duodenal bicarbonate secretion.

BACKGROUND: Exogenous gastrin-releasing peptide (GRP) stimulates the release of secretin from the small intestine and pancreaticobiliary bicarbonate secretion in pigs. As acid is the principal stimulant of secretin release, the purpose of this study was to examine the importance of GRP in acid-induced secretin release and to determine whether GRP contributes to the regulatory function of acid-induced pancreaticobiliary bicarbonate secretion in anaesthetized pigs. METHODS AND RESULTS: Intravenous infusion of GRP (500 pmol/kg.h) increased significantly portal vein plasma concentrations of secretin from 1.3 to 5.4 pmol/l and GRP from 0.5 to 340 pmol/l, pancreatic bicarbonate secretion from 0.01 to 5.9 mmol/h, and hepatic bicarbonate secretion from 0.3 to 3.3 mmol/h, whereas duodenal mucosal bicarbonate secretion remained unchanged. Intravenous infusion of the GRP antagonist BIM-26226 completely abolished the GRP-induced secretin release and pancreatic and hepatic bicarbonate secretion. Furthermore, repeated infusions of GRP did not cause desensitization, and BIM-26226 therefore proved to be an effective GRP antagonist. Duodenal perfusion with acid (pH 1.5, 3.8 mmol/h) significantly increased portal vein plasma concentrations of secretin from 0.4 to 2.8 pmol/l, pancreatic bicarbonate secretion from 0.005 mmol/h to 0.19 mmol/h, hepatic bicarbonate secretion from 0.63 to 2.17 mmol/h, and duodenal mucosal bicarbonate secretion from 0.1 to 1.20 mmol/h. Of importance, infusion of BIM-26226 did not significantly alter the effect of intraduodenal acidification on plasma secretin release and pancreaticobiliary and duodenal bicarbonate secretion. CONCLUSIONS: Thus, we conclude that GRP likely plays an insignificant role in a possible peptidergic regulation of acid-induced intestinal secretin release and that GRP has no regulatory function in acid-induced pancreaticobiliary bicarbonate secretion. Furthermore, GRP has no effect on duodenal bicarbonate secretion.

Gallbladder emptying and cholecystokinin response to fish oil and trioleate ingestion.

The aim of the present study was to compare gallbladder emptying, gastric emptying and release of cholecystokinin (CCK), gastrin and secretin after intragastric administration of fish oil and trioleate. After intravenous injection of 99mTc-HIDA, 30 ml of a lipid labelled with 111In was administered through a gastric tube. Using dual scintigraphy with two markers, gallbladder and gastric emptying were measured simultaneously for 120 min. Plasma concentrations of gastrin, secretin and CCK, were determined throughout the period. The emptying of the gallbladder was reduced by 27% and the release of CCK by 85% after fish oil as compared with trioleate. Gastric emptying as well as the release of gastrin and secretin were similar after the two types of fat. The results suggest that the reduced gallbladder emptying after fish oil may be due to a smaller release of CCK.

Effect of a cholecystokinin (CCK) antagonist (CR 1505) on gene expressions of CCK and secretin in rat intestine.

The effects of intragastric administration of cholecystokinin (CCK) antagonist (CR 1505; 60-300 mg/kg/day) to rats for 3 days on the gene expressions of CCK and secretin, the plasma CCK immunoreactivity, and the CCK content in the intestinal mucosa were examined. CR 1505 increased the level of CCK mRNA in the intestine dose dependently to up to 1.6 times the level in control rats but did not affect the level of secretin mRNA. It also significantly increased the plasma CCK immunoreactivity and the amount of CCK extracted from intestine with acid dose dependently. CR 1505 tended to decrease the trypsin activity in the intestine. These results suggest that ingested CR 1505 increased the CCK mRNA level in the intestine.

Negative feedback regulation of pancreatic exocrine secretion in guinea pigs.

We have investigated whether hormonally mediated negative feedback mechanisms regulate pancreatic exocrine secretion in guinea pigs. In anesthetized guinea pigs prepared with a tube in the proximal duodenum, pyloric ligation, and pancreatic duct cannulation with PE-10 tubing, diversion of pancreatic juice for as long as 4 h in fasting states failed to increase either pancreatic secretion or plasma levels of secretin or cholecystokinin (CCK). In the same animal preparation, intraduodenal (ID) infusion of sodium oleate (SO) resulted in significant increases in both pancreatic secretin and plasma levels of the two hormones that were significantly suppressed by ID infusion of pancreatic juice or a combination of trypsin and chymotrypsin. In another group of guinea pigs, this significant increase in pancreatic secretion was profoundly suppressed by a rabbit antisecretin serum (0.2 ml) or loxiglumide (10 mg.kg-1.h-1). Moreover, a combination of the antiserum and loxiglumide completely abolished the pancreatic secretion. The effect of atropine, 20 micrograms.kg-1.h-1 i.v., on SO-stimulated pancreatic secretion and hormone release was also studied. Atropine completely suppressed the pancreatic secretion of volume flow, bicarbonate, and protein stimulated by SO, whereas neither one of the two hormone levels was affected by intravenous atropine, indicating that atropine blocks the actions of both secretin and CCK on the pancreatic exocrine secretion. It is concluded that a negative feedback regulation of exocrine pancreatic secretion is operative in the intestinal phase of pancreatic secretion in guinea pigs and that this feedback mechanism is mediated by both secretin and CCK. Furthermore, in guinea pigs, cholinergic tone plays an important modulating role in the mechanism.

Effect of intrinsic CCK and CCK antagonist on pancreatic growth and pancreatic enzyme secretion in pancreaticobiliary diversion rats.

When pancreaticobiliary diversion (PBD) surgery was performed in rats, plasma CCK level increased, the pancreas grew mainly by proliferation, and pancreatic trypsinogen showed a remarkable increase, although amylase and lipase synthesis were somewhat decreased. The sensitivity of amylase release against CCK-8 in the pancreatic acini decreased when plasma CCK level was high. These changes in pancreatic growth and pancreatic enzyme secretion caused by PBD were completely inhibited by the CCK-receptor antagonist loxiglumide. From these results, intrinsic CCK was considered to play an important role in both pancreatic enzyme synthesis and proliferation.

Characterization of secretin release in secretin cell-enriched preparation isolated from canine duodenal mucosa.

The release of secretin was studied in secretin cell-enriched preparations isolated from canine duodenal mucosa. The crude enterocytes were isolated by treating the duodenal mucosa sequentially with collagenase and ethylenediaminetetraacetic acid. Secretin cell-enriched fraction was prepared by centrifugation of the crude enterocytes in a counterflow elutriation rotor to obtain a final preparation containing 3.2 +/- 0.3 pmol/10(6) cell of immunoreactive secretin, which was 13-fold greater than the crude cell preparation (N = 5). The cells were incubated in Hanks' balanced salt solution for 20 min at 37 degrees C under 95% O2/5% CO2 before adding various agents and further incubated for various periods of time. The amounts of secretin released into the medium and retained by the cells were then determined by a specific radioimmunoassay. The release of immunoreactive secretin was increased dose-dependently over the control by dibutyryl cyclic-3',5'-adenosine monophosphate, forskolin, 4 beta-12-O-tetradecanoylphorbol-13-acetate, the synthetic serine protease inhibitor, camostat, and the calcium ionophore, A23187. The effects of forskolin, the phorbol ester, and A23187 were time-dependent and not observed at 4 degrees C. The release of immunoreactive secretin was also stimulated by KCl in high concentration and by sodium oleate. The effect of A23187 was abolished in a Ca(2+)-free medium, while those of dibutyryl cyclic-3',5'-adenosine monophosphate and forskolin were potentiated by 3-isobutyl-1-methylxanthine, which did not have a significant effect when added alone. These results indicate that the release of secretin is regulated by both Ca(2+)- and cyclic-3',5'-adenosine monophosphate-dependent mechanisms.2+ release.

The effects of omeprazole on interdigestive motility and early postprandial levels of gastrin and secretin.

Ten healthy men participated in a crossover study, and the experiments took place after 10 days of treatment (40 mg omeprazole every morning). Blood samples were drawn at fixed intervals during a complete migrating motor complex (MMC) cycle. The manometric pressure tube was removed after passage of the second duodenal phase III, and an omelet (1400 KJ) tagged with 99mTc was ingested, followed by 150 ml of water tagged with 111In-diethylenetriaminepentaacetic acid. Mean plasma gastrin (pmol/l) in phases I, II, and III in the omeprazole group was 18.8, 23.3, 19.9, respectively. The corresponding figures for the placebo group were 9.3, 9.6, 9.5, respectively. All mean values for the omeprazole group were significantly higher (p less than 0.01). Mean plasma gastrin in the omeprazole group was significantly higher in phase II than in phase I (p less than 0.05). Mean plasma secretin (pmol/l) in phases I, II, and III in the omeprazole group was 1.6, 1.4, 1.1, respectively. The corresponding figures for the placebo group were 2.0, 1.7, 2.2, respectively. Mean plasma secretin in the omeprazole group was significantly lower in phases I and III (p less than 0.05). The mean incremental integrated postprandial gastrin response (pmol.30 min/l) was significantly higher in the omeprazole group (475.0 versus 97.5) (p less than 0.05). The immediate postprandial mean value of secretin was significantly lower in the omeprazole group (p less than 0.05). We conclude that 40 mg omeprazole elicits i) a phase-related increase in fasting plasma gastrin, ii) a decrease in secretin in phases I and III, iii) an augmented meal-stimulated gastrin response, and iv) a secretin response characterized by a significantly lower mean in the immediate postprandial period.

Plaunotol inhibits postprandial gastrin release by its unique secretin-releasing action in humans.

Plaunotol, an acrylic diterpene alcohol, is a new antiulcer agent derived from the "plau-noi" plant and has been reported to stimulate the release of endogenous secretin in humans. We investigated the effect of plaunotol on postprandial gastrin release, comparing it to the effect of exogenous secretin in a physiological dose in eight healthy volunteers. Four sets of experiments were performed in each volunteer: (1) meal alone, (2) meal after intravenous ranitidine (50 mg), (3) meal after oral administration of plaunotol (320 mg) in addition to ranitidine, and (4) meal after ranitidine with simultaneous intravenous infusion of secretin (0.03 CU/kg/hr). The postprandial increase in plasma secretin concentration was significantly reduced by ranitidine, while postprandial gastrin release was markedly exaggerated. Plaunotol in combination with ranitidine significantly increased secretin release and inhibited gastrin release after a meal. Intravenous infusion of secretin resulted in significant suppression of postprandial gastrin release exaggerated by ranitidine. The present study indicates that plaunotol inhibits postprandial gastrin release by its unique secretin-releasing action.