Regulation of the actin cytoskeleton by p125 focal adhesion kinase in rat pancreatic acinar cells

BACKGROUND/AIMS: Activation of p125 focal adhesion kinase by cholecystokinin (CCK)-8 has recently been demonstrated in pancreatic acinar cells. The purpose of this study is to examine downstream events of this kinase. METHODS: Activation of p125 focal adhesion kinase in freshly isolated rat pancreatic acinar cells was determined by Western blot analysis. Actin cytoskeletal changes were visualized on TRITC-phalloidin-stained cryosections. Amylase release was measured by colorimetric assay. RESULTS: CCK-8 caused dose-dependent activation of p125 focal adhesion kinase. Time-course analysis showed rapid activation with maximum between 5 and 10 min and stimulation still detectable after 60 min. Preincubation with 2 microM cytochalasin D specifically inhibited p125 focal adhesion kinase, but not p42 mitogen-activated protein kinase or increases in intracellular calcium concentrations. The actin cytoskeleton showed rapid reorganization after stimulation, with an initial increase in fluorescence followed by a decline after 30 min. Preincubation with cytochalasin D prevented cytoskeletal changes. Amylase release at concentrations up to 0.1 nM CCK-8 was not influenced by cytochalasin D. In contrast, supramaximal inhibition of amylase release was less pronounced after cytochalasin D incubation. CONCLUSION: p125 focal adhesion kinase in acinar cells appears to be part of a signalling pathway leading to changes in cellular morphology via the actin cytoskeleton. Maximal activation of this signalling pathway might participate in supramaximal inhibition of enzyme secretion.     

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 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.     

Regulation of phospholipid hydrolysis in streptolysin-O-permeabilized rat pancreatic acini.

To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini permeabilized with the bacterial toxin, streptolysin-O. When acini were prelabeled with myo-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+ for 15 min, Ca2+ stimulated [3H]inositol phosphate release at a concentration of 100 nM and was maximally effective at 100 microM. Both GTP gamma S and CCK enhanced Ca(2+)-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+ (100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTP gamma S. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+ over the concentration range from 100 nM to 10 microM. In contrast to PI hydrolysis, however, neither GTP gamma S, CCK, or GTP gamma S plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 microM free Ca2+. Furthermore, Ca(2+)-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.     

Role of guanine nucleotide regulatory protein in stimulation of exocrine pancreatic secretion by cholecystokinin in isolated rat pancreatic acini

To clarify the possible role of a guanine nucleotide regulatory protein in the signal transducing system activated by cholecystokinin (CCK), the actions of CCK on rat pancreatic acini were compared with those of NaF, which is a well-known activator of stimulatory and inhibitory guanine nucleotide regulatory proteins. Guanine nucleotides reduced the binding of 125I-CCK-octapeptide (CCK8) to acinar cell membranes, with the rank order of potency being guanyl-5'-yl imidodiphosphate (Gpp(NH)p) greater than GTP greater than GDP greater than GMP. Scatchard analysis of labeled CCK binding revealed that the decrease in CCK binding caused by Gpp(NH)p was due to the decrease in an affinity constant of CCK for its receptors with no significant change in the maximal binding capacity. When acini were incubated with increasing concentrations of either CCK8 or NaF, maximal stimulation of amylase release occurred at 100 pM CCK8 or 10 mM NaF, respectively, and supramaximal concentrations of CCK8 or NaF caused its submaximal stimulation. Further, CCK and NaF similarly stimulated the hydrolysis of polyphosphoinositide in acini and the release of Ca2+ from the intracellular Ca2+ store into the cytoplasm although there was a lag period prior to any detectable stimulation by NaF. The doses of NaF necessary for Ca2+ mobilization and inositol phosphate generation were nearly the same, with a maximal stimulation at 20 mM NaF. NaF, at concentrations up to 20 mM, a supramaximal concentration for amylase release, produced no significant change in the cellular cyclic AMP level. In addition, 10 mM NaF potentiated the amylase release stimulated by VIP, a well-known secretagogue which functions via cyclic AMP, suggesting that the stimulatory effects of NaF are independent of cellular cyclic AMP. Gpp(NH)p also activated the hydrolysis of polyphosphoinositide in a cell-free pancreatic acinar cell membrane preparation, with a half-maximal and a maximal stimulation at 1 microM and 10 microM, respectively. Furthermore, the effects of submaximal concentrations of CCK8 on polyphosphoinositide hydrolysis were markedly potentiated in the presence of 100 microM GTP, which alone was ineffective. These results, therefore, strongly suggest that pancreatic CCK receptors may be coupled to the activation of polyphosphoinositide breakdown by a guanine nucleotide regulatory protein.     

In vitro and in vivo effect of proglumide on cholecystokinin-stimulated amylase release in mouse pancreatic acini

The effect of proglumide on cholecystokinin (CCK)-stimulated amylase release was studied in vitro and in vivo in dispersed acini from mouse pancreas. In an in vitro study, proglumide at concentrations between 0.3-10 mM inhibited CCK-stimulated amylase release dose-dependently, while proglumide did not influence the basal amylase release at concentrations between 0-3 mM. Dose-response curves to CCK for amylase release shifted to the right with increase in proglumide concentration. This inhibition by proglumide was reversible. In addition, the effect of proglumide was selective for CCK and its related peptide, and this drug did not inhibit other secretagogues such as carbachol or gastrin releasing peptide in mouse acini. In contrast to its inhibitory effect in vitro, in vivo administration of proglumide (500 mg/kg/day, i.p., for 5 days) to mice did not cause the rightward shift of the dose-response curve to CCK for amylase release from dispersed acini.     

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 stellate cells produce acetylcholine and may play a role in pancreatic exocrine secretion

The pancreatic secretagogue cholecystokinin (CCK) is widely thought to stimulate enzyme secretion by acinar cells indirectly via activation of the vagus nerve. We postulate an alternative pathway for CCK-induced pancreatic secretion. We hypothesize that neurally related pancreatic stellate cells (PSCs; located in close proximity to the basolateral aspect of acinar cells) play a regulatory role in pancreatic secretion by serving as an intermediate target for CCK and secreting the neurotransmitter acetylcholine (ACh), which, in turn, stimulates acinar enzyme secretion. To determine whether PSCs (i) exhibit CCK-dependent ACh secretion and (ii) influence acinar enzyme secretion, primary cultures of human and rat PSCs were used. Immunoblotting and/or immunofluorescence was used to detect choline acetyltransferase (ACh synthesizing enzyme), vesicular ACh transporter (VAChT), synaptophysin, and CCK receptors 1 and 2. Synaptic-like vesicles in PSCs were identified by EM. ACh secretion by PSCs exposed to 20 pM CCK was measured by LC-MS/MS. Amylase secretion by acini [pretreated with and without the muscarinic receptor antagonist atropine (10 μM) and cocultured with PSCs] was measured by colorimetry. PSCs express ACh synthesizing enzyme, VAChT, synaptophysin, and CCK receptors; exhibit CCK-dependent ACh secretion; and stimulate amylase secretion by acini, which is blocked by atropine. In conclusion, PSCs express the essential elements for ACh synthesis and secretion. CCK stimulates ACh secretion by PSCs, which, in turn, induces amylase secretion by acini. Therefore, PSCs may represent a previously unrecognized intrapancreatic pathway regulating CCK-induced pancreatic exocrine secretion.     

Involvement of Rab4 in regulated exocytosis of rat pancreatic acini

BACKGROUND & AIMS: Rab4, a Ras-related small guanosine triphosphate (GTP)-binding protein, has been suggested to participate in exocytosis. The function of Rab4 in regulated exocytosis of pancreatic acini was examined in this study. METHODS: Subcellular localization of Rab4 was determined by Western blotting and immunohistochemistry. The Rab4 function in regulated exocytosis was examined by introducing Rab4 hypervariable carboxy-terminal domain peptide (Rab4 peptide) and anti-Rab4 antibody into streptolysin O-permeabilized acini. The regulation of Rab4 by cholecystokinin (CCK) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA) was investigated by examining their effects on [32P]GTP binding rate into the Rab4 immunoprecipitates. The participation of protein kinase C in the Rab4 regulation by CCK was confirmed by calphostin C pretreatment of acini. RESULTS: Rab4 was localized on zymogen granule membranes. Both Rab4 peptide and anti-Rab4 antibody enhanced calcium-stimulated amylase release from streptolysin O-permeabilized acini, suggesting the inhibitory role of Rab4 in exocytosis. CCK and TPA increased GTP binding to Rab4. Calphostin C attenuated the stimulatory effect of CCK on GTP binding to Rab4. CONCLUSIONS: Rab4 negatively modulates regulated exocytosis of pancreatic acini and is controlled by CCK through a protein kinase C pathway.     

Reactive oxygen species activate mitogen-activated protein kinases in pancreatic acinar cells

It has been recently reported that kinases that belong to the mitogen-activated protein kinase (MAPK) family are rapidly activated by cholecystokinin (CCK) in rat pancreas both in vitro and in vivo. It is known that reactive oxygen species (ROS) play an important role in the pathogenesis of acute pancreatitis induced by supraphysiologic stimulation with CCK analogue, cerulein. The aim of our study was to evaluate whether MAPKs are activated by ROS in pancreatic acini. The activity of MAPK, c-Jun amino-terminal kinase (JNK), and p38 MAPK was determined in isolated rat pancreatic acinar cells by means of Western blotting, with the use of specific antibody that recognizes active, dually phosphorylated kinases. Incubation of acini with ROS donors, hydrogen peroxide (H2O2) and/or menadione (MND), strongly activated all three kinases. Activation of these kinases by ROS, but not by CCK, was substantially inhibited by pretreatment of acini with antioxidant N-acetylo-L-cysteine (NAC). Whereas CCK-induced activation of MAPK or JNK was totally or partially blocked by protein kinase C (PKC) inhibitor GF-109203X, ROS-induced activation of MAPK, JNK, and p38 MAPK was PKC independent. In conclusion, ROS strongly activate MAPK, JNK, and p38 MAPK in pancreatic acinar cells. It may be of importance in acute pancreatitis, because ROS are involved in the pathogenesis of this disease.     

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.     

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.     

Effects of a new proglumide analogue CR 1392 on pancreatic exocrine secretion in the rat

The effects of proglumide analogue. CR 1392, on pancreatic exocrine secretion were studied in the isolated pancreatic acini and the isolated perfused pancreata of rats. In the isolated acini, CR 1392 caused a parallel rightward shift of the dose-response curve for amylase secretion stimulated by cholecystokinin octapeptide (CCK-8). CR 1392 inhibited maximally stimulated amylase release by CCK-8 (100 pM) in a concentration-dependent manner, with a half maximal inhibition (ID50) at 8.0 +/- 0.6 microM. CR 1409, another proglumide analogue, also caused a concentration-dependent inhibition (ID50: 3.2 +/- 0.4 microM). Although CR 1409 was about 2.5-fold more potent than CR 1392 in inhibiting the stimulated amylase release, 1 mM CR 1409 caused 107.4 +/- 0.9% increase in amylase release, suggesting acinar cell damage. CR 1392 (1 mM) also caused 19.9 +/- 2.3% increase in amylase release, but was less toxic than CR 1409. The antagonism produced by CR 1392 was selective for CCK and had no effect on amylase release stimulated by other receptor secretagogues or by agents bypassing receptors. CR 1392 added 20 min after the CCK-8 stimulation rapidly abolished pancreatic exocrine secretion in both isolated acini and isolated perfused pancreas. Although the inhibitory effect of CR 1392 was fully reversible in the isolated acini, the pancreata perfused with 100 microM CR 1392 for 20 min did not respond to the subsequent stimulation with CCK-8 for more than 20 min. These results indicate that CR 1392 is a potent, competitive, specific and long acting antagonist of CCK in rat pancreas.     

Cholecystokinin activation of 70-kDa S6 kinase in exocrine pancreas

Cholecystokinin (CCK), a known mitogen for the exocrine pancreas, is shown to activate 70-kDa S6 kinase in isolated pancreatic acini. In this study, we examined the kinetics and cellular mechanisms of CCK-induced p70 S6 kinase activation in vivo and in vitro. Fasted mice were intraperitoneally injected with 0.01–10 g/kg CCK analoge cerulein. Cerulein caused a concentration-dependent activation of p70 S6 kinase, with the maximal effect at 1–10 g/kg. After 1 g/kg cerulein administration, the kinase activity was increased at 5 min, peaked at 10 min, and subsequently decreased. Cerulein also caused a rapid and transient activation of Src. Prior administration of the tyrosine kinase inhibitor herbimycin A compeletely inhibited cerulein-induced Src activation, while the inhibition of p70 S6 kinase activity was partial. Similar results were obtained with pancreatic acinar cell line AR42J cells. These results suggest that tyrosine kinases, including Src as a possible candidate, are partly implicated in the signaling pathway of CCK-induced p70 S6 kinase activation in the exocrine pancreas.     

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.     

Chronic oral administration of synthetic trypsin inhibitor camostate reduces amylase release from isolated rat pancreatic acini

Summary In the present study, we examined stimulus-secretion coupling in pancreatic acini prepared from rats given synthetic protease inhibitor camostate at a dose of 200 mg/kg body wt by an orogastric tube once a day for 10 d. Camostate treatment significantly increased pancreatic weight, protein, DNA, and enzyme contents. In acini prepared from the camostate-treated rats, responsiveness to both CCK-8 and carbamylcholine was greatly decreased with no shift in the dose-response curves compared to control acini prepared from saline-treated rats. There were no major changes in the affinity for both high- and low-affinity sites of CCK receptors, but there was a significant reduction in the capacity of low-affinity site based on acinar protein. Responsiveness to secretin in the camostate-treated rat acini was also significantly reduced compared with that in the controls. However, amylase release from the camostate-treated rat acini in response to an increase in intracellular calcium levels induced by the calcium ionophores A23187 or to an increase in intracellular cyclic 3′,5′-monophosphate (cyclic AMP) levels caused by 8 bromo cyclic AMP was not significantly different from the control rat acini, suggesting that both Ca²⁺-dependent tyrosine kinase and nucleotide-activated kinases are not impaired. On the other hand, the responsiveness to phorbol ester TPA, which stimulates amylase secretion via a calcium-independent cascade by activating protein kinase C directly, was reduced in the camostate-treated rat acini compared with the controls. These results suggest the possibilities that the reduced amylase secretion in the camostate-treated rats is owing to alterations in both the transmembrane signal transduction and the phosphorylation of regulatory proteins by the Ca²⁺-independent, protein kinase C-dependent mechanisms.     

Impaired pancreatic exocrine function in rats with carbon tetrachloride-induced liver cirrhosis

Summary Background Substantial numbers of studies have revealed the close correlation between chronic pancreatitis and cirrhosis in human. However, the situation with regard to pancreatic enzyme secretion is less clear. Aim The aim of the study was to investigate pancreatic exocrine function in rat with carbon tetrachloride-induced liver cirrhosis in rats. Methods Pancreatic exocrine function and morphology in Sprague-Dawley rats with carbon tetrachloride-induced liver cirrhosis were investigated. Pancreatic exocrine functions stimulated by cholecystokinin-8 and other secretagogs were assessed in isolated pancratic acini, and in vivo and morphological changes were studied by routine were assessed in isolated pancreatic acini, and in vivo and morphological changes were studied by routine histological examination and electron microscopy. Results The basal and cholecystokinin-8-stimulated amylase releases from acini and acinar amylase content were significantly lower in the cirrhotic rats than the control. None of the secretagogs induced the some amount of amylase release in cirrhotic as in control rats. Volume of the pancreatic juice and outputs of amylase and protein were significantly decreased under basal and cholecystokinin-8-stimulated conditions in vivo. Electron microscopy revealed most of the rough-surfaced endoplasmic reticulum accompanying less numbers of ribosomes to be dilated and some mitochondria to be swollen in cirrhotic rats. Conclusion Pancreatic exocrine functions are decreased in cirrhotic rats owing to alterations at the electron microscopic levels, reflecting an impaired acinar intracellular messenger system.     

Pharmacologic profile of TS-941, a new benzodiazepine derivative cholecystokinin-receptor antagonist, in in vitro isolated rat pancreatic acini

We investigated the pharmacologic characteristics of a newly developed benzodiazepine derivative (S)-(-)-N-[2,3-dihydro-2-oxo-5-phenyl-1-[(1H-tetrazol-5-yl)methyl] -1H-1,4-benzodiazepine-3-yl]-2-indolecarboxamide (TS-941), a cholecystokinin type A (CCK-A)-receptor antagonist, in the isolated rat pancreatic acini and compared with those of well-known CCK-A-receptor antagonists, devazepide and loxiglumide. TS-941 inhibited CCK-8-stimulated amylase release concentration dependently, as did devazepide and loxiglumide, with a half-maximal inhibition (IC50) at 78.6 +/- 10.3 nM. TS-941 was approximately 23 times less potent than devazepide (IC50, 3.4 +/- 0.3 nM), but was 50 times more potent than loxiglumide (IC50, 3,966 +/- 544 nM) in inhibiting 100 pM CCK-8-stimulated amylase release from rat pancreatic acini. TS-941 had a fivefold lower selectivity than devazepide for pancreatic CCK (CCK-A) over brain CCK (CCK-B) receptors but fourfold greater than loxiglumide when IC50 values for inhibition of [125I]CCK-8 binding in isolated acini and cerebral cortex were compared. The antagonism produced by TS-941 was specific for CCK in that the effects of other receptor secretagogues or agents bypassing receptors were not altered. TS-941 caused a parallel rightward shift of the entire dose-response curve for CCK-8-stimulated amylase release without altering the maximal increase, as did devazepide and loxiglumide. TS-941, whether added at the beginning or 20 min after the CCK-8 stimulation, inhibited amylase release. TS-941 caused a concentration-dependent residual inhibition of the action of CCK-8. The acini, once incubated with a high concentration of TS-941 (10 microM; 127 times IC50) for 30 min, was 10-fold less sensitive to CCK-8 than the acini preincubated without TS-941, whereas the sensitivity and the responsiveness to CCK-8 stimulation of those incubated with a low concentration of TS-941 (1.0 microM) were similar to the control acini. These results indicate that TS-941 is a potent, competitive, and selective CCK-A receptor antagonist for the pancreas.     

Inositol 1,4,5-trisphosphate formation, cytoplasmic calcium dynamics, and alpha-amylase secretion of pancreatic acini isolated from aged and chronically alcohol-fed rats.

METHODS: Three-month-old female Wistar rats were fed with 20% alcohol in their drinking fluid over 6-17 mo using an interrupted feeding regimen. At different times, pancreatic acini were isolated by mild collagenase digestion. The concentrations of inositol-1,4,5-trisphosphate (1,4,5-IP3) were determined by a specific radioreceptor assay, before and at different times after stimulation with varying concentrations of CCK-8. CCK-induced dynamics of cytoplasmic calcium ([Ca2+]c) was investigated in acinar cells by confocal laser raster microscopy. Acinar alpha-amylase (Aml) secretion was measured as enzyme activity in the medium compared to the total activity in the suspension. RESULTS: In 12-13-mo-old rats, the CCK-stimulated 1,4,5-IP3 formation in acini was found to be decreased compared to young rats (age 4 mo). In rats of the same age fed with ethanol from the age of 3 mo on, 1,4,5-IP3 concentrations in acini were higher and reached values comparable to those in young rats. Correspondingly, the CCK-induced [Ca2+]c dynamics in acini isolated from 9-mo-old rats was impaired compared to that of young rats but normal in aged, chronically alcohol-fed rats. Aml secretion under CCK stimulation, however, which was decreased in aged rats, was additionally impaired after alcohol feeding. CONCLUSION: Chronic alcohol feeding modifies 1,4,5-IP3 formation, the [Ca2+]c dynamics of, and the Aml secretion of rat pancreatic acini in response to CCK stimulation. Obviously, the age-related impairment of 1,4,5-IP3 formation and [Ca2+]c dynamics is improved. In contrast, the decrease in Aml secretion of acini isolated from aged rats is more pronounced after long-term alcohol-feeding.     

Effect of hydrocortisone on the maturation of cholecystokinin (CCK) binding and CCK stimulated amylase release in pancreatic acini of neonatal rats

Neonatal rat pancreata are not responsive to stimulation by cholecystokinin (CCK) and this has been shown to be due primarily to low binding of CCK to pancreatic acinar cells of rats of this age group. To see if hydrocortisone has any effect on the maturation of CCK binding and enzyme secretion, day-old rat pups were injected three times intraperintoneally with hydrocortisone at a dose of 5 mg/100 g body weight per dose and sacrificed 48 h after the first injection. Control age-matched pups were injected with 0.9% saline at the same volume and schedule as the hydrocortisone injected pups. The pancreatic weight, protein, and DNA contents were found to be significantly lower in the pups from the hydrocortisone-treated group than in the pups from the control group. The protein content per unit weight of DNA, however, was not different between the two. The maximal output of amylase under stimulation by 3 X 10(-10) M CCK was significantly higher in the dispersed acini prepared from the hydrocortisone-treated group as compared to dispersed acini prepared from the control (575 +/- 50 vs. 390 +/- 40% when expressed as a percentage of basal release). The maximal binding to 125I-BH-CCK was also significantly higher in the dispersed acini from the hydrocortisone group when compared to the dispersed acini from the control group (2.6 +/- 0.5 vs. 1.4 +/- 0.4%). Hydrocortisone, therefore, induces the precocious maturation of the secretory apparatus of the pancreatic acini, specifically the increase in capacity to bind and the greater responsiveness of the acini to CCK.