Effect of Tetrandrine on LPS-induced NF-κB activation in isolated pancreatic acinar cells of rat

AIM: To investigate the effect of Tetrandrine (Tet) on LPS-induced NF-κB activation and cell injury in pancreatic acinar cells and to explore the mechanism of Tetrandrine preventing LPS-induced acinar cell injury. METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to LPS (10 mg/L), Tet (50μmol/L, 100μmol/L) or normal media. At different time point (30 min, 1 h, 4 h, 10 h) after treatment with the agents, cell viability was determined by MTT, the product and nuclear translocation of subunit p65 of NF-κB was visualized by immunofluorescence staining and nuclear protein was extracted to perform EMSA which was used to assay the NF-κB binding activity. RESULTS: LPS induced cell damage directly in a time dependent manner and Tet attenuated LPS-induced cell damage (50μmol/L, P < 0.05; 100μmol/L, P < 0.01). NF-κB p65 immunofluorescence staining in cytoplasm increased and began showing its nuclear translocation within 30 min and the peak was shown at 1 h of LPS 10 mg/L treatment. NF-κB DNA binding activity showed the same alteration pattern as p65 immunofluorescence staining. In Tet group, the immunofluorescence staining in cytoplasm and nuclear translocation of NF-κB were inhibited significantly. CONCLUSION: NF-κB activation is an important early event that may contribute to inflammatory responses and cell injury in pancreatic acinar cells. Tet possesses the protective effect on LPS-induced acinar cell injury by inhibiting NF-κB activation.     

The pre—synaptic blocker toosendanin does not inhitit secretion in exocrine cells

AIM：Toosendanin is a pre-synaptic blocer at the neuromuscular junction and its inhititory effect is divided into an initial facilitatve/stimulatory phase followed by a prolonged inhibitory phase,The present study investigated whether the subsequent inhibitory phase was due to exhaustion of the secretory machinery as a result of extensive stimulation during the initial facilitative phase More specifically,this paper examined whether toosendanin could directly inhibit the secretory machinery in exocrine cells.METHODS:Rat pancreatic acinar cells were isolated by collagenase digestion,Secretion was assessed by measuring the amount of amylase released into the extracellular medium as a percentage of the total present in the cells before stimulation.Cholecystokinin(CCK)-induced increases in intracellular calcium in single cells were measured with fura-2microfluorometry.RESULTS:Effects of toosendanin on CCK-induced amylase secretion and calcium oscillations were investigated.Toosendanin of 87-870μMhad no effect on 10pM-100nMCCK-stimulated amylase secretion.nor did 8.7-870μMtoosendanin inhibit 5pM CCK-induced calcium oscillations.In contrast,10nMCCK1recepto antagonistFK480completely blocked5pM CCK-induced calcium oscillations.CONCLUSION;The pre-synaptic“blocker”toosendanin is a selective activator of the voltage-dependent calcium channels but does not interfere with the secretory machinery itself.     

Pancreatic regenerating protein (regⅠ) and regⅠreceptor mRNA are upregulated in rat pancreas after induction of acute pancreatitis

AIM: Pancreatic regenerating protein (regⅠ) stimulates pancreatic regeneration after pancreatectomy and is mitogenic to ductal andβ-cells. This suggests that regⅠand its receptor may play a role in recovery after pancreatic injury. We hypothesized that regⅠand its receptor are induced in acute pancreatitis. METHODS: Acute pancreatitis was induced in male Wistar rats by retrograde injection of 3% sodium taurocholate into the pancreatic duct. Pancreata and serum were collected 12, 24, and 36 hours after injection and from normal controls (4 rats/group). RegⅠreceptor mRNA, serum regⅠprotein, and tissue regⅠprotein levels were determined by Northern analysis, enzyme-linked immunosorbent assay (ELISA), and Western analysis, respectively. Immunohistochemistry was used to localize changes in regⅠand its receptor. RESULTS: Serum amylase levels and histology confirmed necrotizing pancreatitis in taurocholate treated rats. There was no statistically significant change in serum regⅠconcentrations from controls. However, Western blot demonstrated increased tissue levels of regⅠat 24 and 36 h. This increase was localized primarily to the acinar cells and the ductal cells by immunohistochemistry. Northern blot demonstrated a significant increase in regⅠreceptor mRNA expression with pancreatitis. Immunohistochemistry localized this increase to the ductal cells, islets, and acinar cells. CONCLUSION: Acute pancreatitis results in increased tissue regⅠprotein levels localized to the acinar and ductal cells, and a parallel threefold induction of regⅠreceptor in the ductal cells, islets, and acinar cells. These changes suggest that induction of reg I and its receptor may be important for recovery from acute pancreatitis.     

Alteration of chaperonin60 and pancreatic enzyme in pancreatic acinar cell under pathological condition

AIM: To investigate the changes of chaperonin60 (Cpn60) and pancreatic enzymes in pancreatic acinar cells, and to explore their roles in the development of experimental diabetes and acute pancreatitis (AP). METHODS: Two different pathological models were replicated in Sprague-Dawley rats: streptozotocin-induced diabetes and sodium deoxycholate-induced AP. The contents of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were measured by quantitative immunocytochemistry. RESULTS: The levels of Cpn60 significantly increased in diabetes, but decreased in AP, especially in the zymogen granules of the pancreatic acinar cells. The elevation of Cpn60 was accompanied with the increased levels of pancreatic lipase and chymotrypsinogen in diabetes. However, a decreased Cpn60 level was accompanied by high levels of lipase and chymotrypsinogen in AP. The amylase level was markedly reduced in both the pathological conditions. CONCLUSION: The equilibrium between Cpn60 and pancreatic enzymes in the acinar cells breaks in AP, and Cpn60 content decreases, suggesting an insufficient chaperone capacity. This may promote the aggregation and autoactivation of the premature enzymes in the pancreatic acinar cells and play roles in the development of AP.     

Signal pathways involved in emodin-induced contraction of smooth muscle cells from rat colon

AIM: To investigate the effects induced by emodin on single smooth muscle cells from rat colon in vitro, and to determine the signal pathways involved.METHODS: Cells were isolated from the muscle layers of Wistar rat colon by enzymatic digestion. Cell length was measured by computerized image micrometry. Intracellular Ca^2+ ([Ca^2+]i) signals were studied using the fluorescent Ca^2+ indicator fluo-3 and confocal microscopy. PKCα distribution at rest state or after stimulation was measured with immunofluorescence confocal microscopy.RESULTS: (1) Emodin dose-dependently caused colonic smooth muscle cells contraction, (2) emodin induced an increase in intracellular Ca^2+ concentration; (3) the contractile responses induced by emodin were respectively inhibited by preincubation of the cells with ML-7 (an inhibitor of MLCK) and calphostin C (an inhibitor of PKC), (4) Incubation of cells with emodin caused translocation of PKCα from cytosolic area to the membrane.CONCLUSION: Emodin has a direct contractile effect on colonic smooth muscle cell. This signal cascade induced by emodin is initiated by increased [Ca^2+]i and PKCα translocation,which in turn lead to the activation of MLCK and the suppression of MLCP. Both of them contribute to the emodininduced contraction.     

Pathophysiology of chronic pancreatitis induced by dibutyltin dichloride joint ethanol in mice

AIM: To search for a new chronic pancreatitis model in mice suitable for investigating the pathophysiological processes leading to pancreatic fibrosis.METHODS: The mice were randomly divided into 2 groups(n = 50), control group and model group. The mice in model group were given ethanol(10%) in drinking water after injection of dibutyltin dichloride(DBTC)(8 mg/kg BW) in tail vein. The mice in control group were injected with only solvent into tail vein( 60 % ethanol, 20% glycerine and 20% normal saline) and drank common water. At days 1, 7, 14, 28, and 56 after application of DBTC or solvent, 10 mice in one group were killed at each time point respectively. Blood was obtained by inferior vena cava puncture. The activity of amylase, concentration of bilirubin and hyaluronic acid in serum were assayed. The pancreas was taken to observe the pancreatic morphology by HE staining, and to characterize the pancreatic fibrosis by Masson staining. The expression of F4/80, CD3 and fibronectin(FN) were assayed by immuno-histochemistry or Immunofluorescence technique. Collagen typeⅠ(COL1A1) in pancreas were detected by Western blot. The expression of matrix metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinases-1(TIMP-1) m RNA in the pancreas was assessed by real time PCR.RESULTS: DBTC induced an acute edematous pancreatitis within 1 d. The dilated acini, scattered acinar cell necrosis, and inflammatory cells were found at day 7. Extensive infiltration with inflammatory cells following deposition of connective tissue was observed at day 14. At day 28, level of pancreatic fibrosis was aggravated. The pancreatic tissue was replaced by an extended interstitial fibrosis at the end of 2 mo. There was significant difference in the level of amylase, bilirubin and hyaluronic acid in serum between control group and model group(P 0.05). The level of COL1A1 and FN in pancreas increased. The expression of MMP-1 m RNA in pancreas decreased, but TIMP-1 m RNA increased at model group.CONCLUSION: DBTC joint Ethanol drinking can induce chronic pancreatitis in accordance with the pathophysiological modification of human. DBTC joint Ethanol-induced pancreatitis in mice is an effective and handy experimental method. The model is suitable to study the mechanism of pancreatic fibrosis in chronic pancreatitis.     

Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis

Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy.The pathogenesis of pancreatitis is still not well understood.Calcium(Ca2+)is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells.Ca2+overload is a key early event and is crucial in the pathogenesis of many diseases.In pancreatic acinar cells,pathological Ca2+signaling(stimulated by bile,alcohol metabolites and othercauses)is a key contributor to the initiation of cell injury due to prolonged and global Ca2+elevation that results in trypsin activation,vacuolization and necrosis,all of which are crucial in the development of pancreatitis.Increased release of Ca2+from stores in the intracellular endoplasmic reticulum and/or increased Ca2+entry through the plasma membrane are causes of such cell damage.Failed mitochondrial adenosine triphosphate(ATP)production reduces re-uptake and extrusion of Ca2+by the sarco/endoplasmic reticulum Ca2+-activated ATPase and plasma membrane Ca2+-ATPase pumps,which contribute to Ca2+overload.Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca2+signals in pancreatitis.The lack of available specific treatments is therefore an objective of ongoing research.Research is currently underway to establish the mechanisms and interactions of Ca2+signals in the pathogenesis of pancreatitis.     

Mechanism for Src activation by the CCK2 receptor： Pathophysiological functions of this receptor in pancreas

AIM: To investigate in vivo, whether CCK2 receptors (CCK2R) regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation. METHODS: The expression and activation of Src and ERK were studied in vivo using immuno-fluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or Elas-CCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation. Src activation was measured by in vitro kinase assays, ERK activation by western blot using anti-phospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test. RESULTS: We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7th transmembrane domain, and suggested the putative role of Gq in this mechanism. CONCLUSION: Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development.     

Polyethylene glycol 35 ameliorates pancreatic inflammatory response in cerulein-induced acute pancreatitis in rats

BACKGROUND Acute pancreatitis(AP) is a sudden inflammatory process of the pancreas that may also involve surrounding tissues and/or remote organs. Inflammation and parenchymal cell death are common pathological features of this condition and determinants of disease severity. Polyethylene glycols(PEGs) are nonimmunogenic, non-toxic water-soluble polymers widely used in biological, chemical, clinical and pharmaceutical settings.AIM To evaluate the protective effect of a 35-k Da molecular weight PEG(PEG35) on the pancreatic damage associated to cerulein-induced acute pancreatitis in vivo and in vitro.METHODS Wistar rats were assigned at random to a control group, a cerulein–induced AP group and a PEG35 treatment group. AP was induced by five hourly intraperitoneal injections of cerulein(50 μg/kg/bw), while the control animals received saline solution. PEG35 was administered intraperitoneally 10 minutes before each cerulein injection in a dose of 10 mg/kg. After AP induction, samples of pancreatic tissue and blood were collected for analysis. AR42 J pancreatic acinar cells were treated with increasing concentrations of PEG35 prior to exposure with tumor necrosis factor α(TNFα), staurosporine or cerulein. The severity of AP wasdetermined on the basis of plasma levels of lipase, lactate dehydrogenase activity, pancreatic edema and histological changes. To evaluate the extent of the inflammatory response, the gene expression of inflammation-associated markers was determined in the pancreas and in AR42 J-treated cells. Inflammation-induced cell death was also measured in models of in vivo and in vitro pancreatic damage.RESULTS Administration of PEG35 significantly improved pancreatic damage through reduction on lipase levels and tissue edema in cerulein-induced AP rats. The increased associated inflammatory response caused by cerulein administration was attenuated by a decrease in the gene expression of inflammation-related cytokines and inducible nitric oxide synthase enzyme in the pancreas. In contrast, pancreatic tissue m RNA expression of interleukin 10 was markedly increased. PEG35 treatment also protected against inflammation-induced cell death by attenuating lactate dehydrogenase activity and modulating the pancreatic levels of apoptosis regulator protein BCL-2 in cerulein hyperstimulated rats. Furthermore, the activation of pro-inflammatory markers and inflammationinduced cell death in pancreatic acinar cells treated with TNFα, cerulein or staurosporine was significantly reduced by PEG35 treatment, in a dose-dependent manner.CONCLUSION PEG35 ameliorates pancreatic damage in cerulein-induced AP and AR42 J-treated cells through the attenuation of the inflammatory response and associated cell death. PEG35 may be a valuable option in the management of AP.     

Munc18/SNARE proteins’ regulation of exocytosis in guinea pig duodenal Brunner’s gland acini

AIM： To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS： We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS： One and 6 μmol/L carbachol evoked a dose-dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION： Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.     

Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum.

The effects of extracellular phosphate and lanthanum on cytosolic free Ca2+ [( Ca2+]i) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca2+ and 1.0 mM phosphate, the mean resting [Ca2+]i level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both [Ca2+]i levels and 45Ca2+ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca2+ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated [Ca2+]i levels were very rapid and were mimicked only by prolonged incubation of acini in Ca2+-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal [Ca2+]i levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting [Ca2+]i levels in pancreatic acini and other cell types and that mobilization of intracellular Ca2+ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca2+ that is not readily removed by EGTA.     

Effects of phospholipase A2 inhibitors on Ca2+ oscillations in pancreatic acinar cells

High-affinity cholecystokinin (CCK) receptors were reported to be coupled with phospholipase A2 (PLA2)-arachidonic acid (AA) pathways to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells. To investigate which types of PLA2 were involved in PLA2-AA pathways, the effects of specific inhibitors for type II and type IV PLA2 on Ca2+ oscillations and amylase secretion were studied in isolated rat pancreatic acini. An inhibitor of type IV (cytosolic) PLA2, AACOCF3 inhibited Ca2+ oscillations elicited by CCK-8 (30 pM) and JMV-180 (100 nM). AACOCF3 inhibited amylase secretion stimulated by JMV-180 and low concentrations of CCK-8 (< or =30 pM). On the other hand, an inhibitor of type II (secretory, nonpancreatic) PLA2 had no effects on Ca2+ oscillations and amylase secretion stimulated by CCK-8 and JMV-180. These results suggest that high-affinity CCK receptors are coupled to cytosolic PLA2 to mediate Ca2+ oscillations and amylase secretion in rat pancreatic acinar cells.     

Distribution of nitric oxide synthase and secretory role of exogenous nitric oxide in the isolated rat pancreas.

BACKGROUND: Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. METHODS: NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method. Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca2+]i) was measured in dispersed pancreatic acinar cells. RESULTS: NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca2+]i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca2+]i. CONCLUSION: NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nerve terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.     

Effect of ethanol on pancreatic exocrine secretion in rats.

The effect of ethanol on pancreatic exocrine secretion was studied in isolated rat pancreatic acini. Ethanol caused a dose-dependent stimulation of amylase release, and a twofold increase of amylase release was observed with 600 mM ethanol. Ethanol inhibited cholecystokinin octapeptide (CCK-8)- and carbamylcholine-stimulated amylase release and similarly inhibited binding of [125I]CCK-8 and [N-methyl-3H]scopolamine to isolated rat pancreatic acini in a dose-dependent manner. The inhibitory effect of ethanol was fully reversible with respect to CCK-8-induced amylase release. On the other hand, ethanol potentiated secretin- and vasoactive intestinal peptide (VIP)-stimulated amylase release. Ethanol induced a small but significant increase in Ca2+ efflux, whereas CCK-8 induced an immediate and large increase, but ethanol significantly inhibited CCK-8-stimulated Ca2+ efflux. The present study clearly demonstrates the dual effects of ethanol on pancreatic exocrine function: stimulation and inhibition. We suggest that mobilization of intracellular Ca2+ may be involved in the mechanism of ethanol's action on isolated rat pancreatic acini.     

Free cytosolic calcium and secretagogue-stimulated initial pancreatic exocrine secretion

In order to establish the role of secretagogue-induced changes in free cytosolic Ca2+ ([Ca2+]i) in pancreatic enzyme secretion, we measured the effects of carbachol, cholecystokinin-octapeptide (CCK-OP), bombesin, substance P, and bromo-A23187 on amylase release and [Ca2+]i in guinea pig pancreatic acini loaded with the Ca2+-selective fluorescent indicator, fura-2. Evaluation of time courses and dose-response curves indicated that carbachol, CCK-OP, bombesin, and substance P cause extracellular Ca2+-independent transient increases in [Ca2+]i and transient bursts in amylase release (initial secretion). The potencies for the secretagogues to increase [Ca2+]i and initial amylase release were similar. Bromo-A23187 also caused an extracellular Ca2+-independent transient increase in [Ca2+]i and amylase release. In the absence of extracellular Ca2+, sequential additions of substance P followed by carbachol caused transient increases in [Ca2+]i correlating with transient bursts in amylase release. In contrast, in acini first treated with carbachol, the ability of substance P to increase [Ca2+]i and amylase release was blocked. Sustained secretion caused by the secretagogues was dependent on extracellular Ca2+ but occurred at basal [Ca2+]i. Increasing [Ca2+]i during the sustained phase of stimulation by increasing the extracellular Ca2+ concentration or with bromo-A23187 did not increase the rate of sustained secretion.     

Effects of calmodulin inhibitors on amylase secretion from rat pancreatic acini.

To investigate the role of calmodulin in stimulus-secretion coupling in pancreatic acinar cells, we studied the effects of W-7, a calmodulin inhibitor, and KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II), on amylase secretion from rat pancreatic acini. Calmodulin inhibitor (W-7, 100 microM) and Ca2+/CaM kinase II inhibitor (KN-62, 10 microM) reduced amylase secretion stimulated by cholecystokinin (CCK) or carbachol. W-7 and KN-62 also inhibited amylase secretion stimulated by both calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). To clarify the role of calmodulin in the interaction of intracellular mediators, pancreatic acini were permeabilized with streptolysin O. Following permeabilization, amylase secretion was stimulated by submicromolar free Ca2+, and this Ca(2+)-dependent amylase secretion was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), TPA or cyclic adenosine 3',5'-monophosphate (cAMP). W-7 and KN-62 had no effects on amylase secretion stimulated by Ca2+ alone, but inhibited the enhancement in Ca(2+)-dependent amylase secretion by GTP gamma S, TPA or cAMP. These data suggest that calmodulin plays an important role in Ca(2+)-dependent amylase secretion from pancreatic acinar cells and in the interaction between Ca2+ and other intracellular messengers.     

Effect of somatostatin on cholecystokinin-induced amylase release in rat pancreatic acini.

The effect of somatostatin on cholecystokinin-induced amylase release was investigated in isolated rat pancreatic acini. Acini were isolated by enzymatic digestion and incubated in a HEPES buffered Ringer's solution with testing reagents for 30 minutes at 37 degrees C. The activity of released amylase, cAMP, and inositol phosphate formation were measured. Intracellular calcium concentration ([Ca2+]i) was also checked. Somatostatin 14 and octreotide, a somatostatin analog, inhibited CCK-stimulated amylase release in a concentration-dependent manner. The inhibitory effect of octreotide on CCK-induced amylase release was not shown when the acini were treated with 8-Br-cAMP, irrespective of the presence of IBMX. Forskolin potentiated CCK-induced amylase release and this effect was blocked by octreotide treatment; although CCK-8 (3 x 10(-11) M) failed to stimulate cAMP formation, octreotide significantly inhibited basal cAMP formation in the acini. The increase of [Ca2+]i in response to CCK was inhibited by octreotide. However, CCK-induced inositol phosphate formation was not changed by 10(-9) M octreotide. Octreotide had no effect on CCK-stimulated tyrosine phosphorylation, and tyrosine phosphatase inhibitors (NaF and Na2WO4) did not influence the effect of octreotide on CCK-induced amylase release. From these results, we conclude that octreotide inhibits CCK-induced amylase release by inhibiting basal cAMP formation and decreasing the [Ca2+]i stimulated by CCK.     

Effects of L-364,718 on pancreatic exocrine and endocrine secretion in the rat.

We examined the inhibitory effect of L-364,718, a nonpeptide cholecystokinin (CCK) antagonist, on CCK stimulation of pancreatic exocrine and endocrine secretion in both the isolated pancreatic acini and the isolated perfused pancreata of rats. In the isolated acini, L-364,718 inhibited CCK octapeptide (CCK-8)-stimulated amylase release and binding of 125I-CCK-8 in a dose-dependent manner without appreciable effects on the basal amylase secretion. L-364,718 also inhibited amylase release in response to caerulein and gastrin I, but had no effect on amylase release stimulated by other secretagogues or by agents bypassing receptors. Similarly, binding of N-methylscopolamine to pancreatic acini was not inhibited by L-364,718. In the isolated perfused pancreata, L-364,718 inhibited CCK-8-stimulated pancreatic exocrine secretion and insulin release. The inhibitory effects of L-364,718 were more potent for insulin release than for exocrine secretion and persisted even after the removal of L-364,718 infusion. These results clearly demonstrate that L-364,718 is a specific, potent, and prolonged antagonist of CCK's stimulatory actions on pancreatic acinar and B cells.     

Metalloendopeptidase inhibitors and stimulus-secretion coupling in the mouse exocrine pancreas.

Inhibitors of metalloendopeptidases interfere with events involving Ca2(+)-dependent membrane fusion in a number of cell types. The divalent ion chelating agent 1,10-phenanthroline inhibited pancreatic amylase secretion stimulated by carbachol, cholecystokinin-octapeptide (CCK-8), or bombesin, but detailed studies indicated that this is unlikely to be a result of inhibition of metalloendopeptidase activity. The binding of [3H]N-methylscopolamine to pancreatic acini was reduced by 1,10-phenanthroline and this would explain the marked inhibition of carbachol-induced amylase secretion by the chelating agent. CCK-8-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate was reduced by 1,10-phenanthroline while the binding of CCK-8 to acini was not affected. This inhibition of hydrolysis would explain the inhibition of CCK-8- and bombesin-induced amylase secretion. The metalloendopeptidase substrate carbobenzoxyglycylphenylalanylamide did not affect bombesin-stimulated amylase secretion. Amylase secretion evoked by treating pancreatic acini with the ionophore A23187 or dibutyryl-cyclic AMP was not reduced by 1,10-phenanthroline, indicating a lack of involvement of metalloendopeptidases in the process of exocytosis in this cell type.     

A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.