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In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3β pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells., (Published by Elsevier B.V.)

P-21-activated kinases (PAKs) are serine/threonine kinases comprising six isoforms divided in two groups, group-I (PAK1-3)/group-II (PAK4-6) which play important roles in cell cytoskeletal dynamics, survival, secretion and proliferation and are activated by diverse stimuli. However, little is known about PAKs ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth-factors. We used rat pancreatic acini to explore the ability of GI-hormones/neurotransmitters/growth-factors to activate Group-I-PAKs and the signaling cascades involved. Only PAK2 was present in acini. PAK2 was activated by some pancreatic growth-factors [EGF, PDGF, bFGF], by secretagogues activating phospholipase-C (PLC) [CCK, carbachol, bombesin] and by post-receptor stimulants activating PKC [TPA], but not agents only mobilizing cellular calcium or increasing cyclic AMP. CCK-activation of PAK2 required both high- and low-affinity-CCK1-receptor-state activation. It was partially reduced by PKC- or Src-inhibition, but not with PI3K-inhibitors (wortmannin, LY294002) or thapsigargin. IPA-3, which prevents PAK2 binding to small-GTPases partially inhibited PAK2-activation, as well as reduced CCK-induced ERK1/2 activation and amylase release induced by CCK or bombesin. This study demonstrates pancreatic acini, possess only one Group-I-PAK, PAK2. CCK and other GI-hormones/neurotransmitters/growth-factors activate PAK2 via small GTPases (CDC42/Rac1), PKC and SFK but not cytosolic calcium or PI3K. CCK-activation of PAK2 showed several novel features being dependent on both receptor-activation states, having PLC- and PKC-dependent/independent components and small-GTPase-dependent/independent components. These results show that PAK2 is important in signaling cascades activated by numerous pancreatic stimuli which mediate their various physiological/pathophysiological responses and thus could be a promising target for the development of therapies in some pancreatic disorders such as pancreatitis., (Copyright © 2015. Published by Elsevier B.V.)

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects., (Published by Elsevier B.V.)

The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth)., (Published by Elsevier B.V.)

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.

The receptor of hepatocyte growth factor (HGF), c-met induces different physiological responses in several cell types. Little is known about the role of HGF in exocrine pancreas. However, abnormal HGF signaling has been strongly implicated in pancreatic tumorigenesis and association of HGF with pancreatitis has been demonstrated. We have studied the presence of c-met and activation of their intracellular pathways associated in rat pancreatic acini in comparison with cholecystokinin (CCK) and epidermal growth factor (EGF). C-met expression in rat exocrine pancreas was identified by immunohistochemistry and immunoprecipitation followed by Western analysis. Tyrosine phosphorylation of c-met is strongly stimulated as well as kinase pathways leading to ERK1/2 cascade. HGF, but not CCK or EGF, selectively caused a consistent increase in the amount of p85 regulatory subunit of PI3-K present in anti-phosphotyrosine immunoprecipitates. Downstream of PI3-K, HGF increased Ser473 phosphorylation of Akt selectively, as CCK or EGF did not affect it. HGF selectively stimulated tyrosine phosphorylation of phosphatase PTP1D. HGF failed to promote the well-known CCK effects in pancreatic acini such as amylase secretion and intracellular calcium mobilization. Although HGF shares activation of ERK1/2 with CCK, we demonstrate that it promotes the selective activation of intracellular pathways not regulated by CCK or EGF. Our results suggest that HGF is an in vivo stimulus of pancreatic acini and provide novel insight into the transduction pathways and effects of c-met/HGF in normal pancreatic acinar cells.

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin.

The actions of neuromedin B (NMB), a recently discovered mammalian bombesin-related peptide, are mediated by interacting with a distinct receptor; however, little is known about its cellular basis of action. Recent studies show activation of phospholipase D (PLD) is an important transduction cascade for a number of GI hormones, especially for stimulation of growth and protein sorting. The purpose of the present study was to determine whether activation of the NMB receptor causes activation of PLD and to explore whether this activation was coupled to PLC activation. Rat C6 glioblastoma cells (C6 cells), which contain a low density of native NMB receptors and BALB 3T3 cells stably transfected with rat NMB receptors, were used. NMB caused a 3-fold increase in C6 cells and an 11-fold increase in rNMB-R transfected cells in PLD activity. Increases in PLD activity were rapid and NMB was 100-fold more potent than gastrin-releasing peptide (GRP). NMB caused a half-maximal increase in [Ca2+]i at 0.2 nM, in [3H]IP and PLD at 1 nM, and half-maximal receptor occupation at 1.2 nM. TPA increased PLD dose-dependently with a half-maximal effect at 60 nM. The calcium ionophore A23187 (1 microM) alone did not increase PLD activity but potentiated the effect of TPA. The Ca2+-ATPase inhibitor, thapsigargin, did not affect NMB- or TPA-stimulated PLD activities, although it blocked completely the NMB-induced increase in [Ca2+]i. The PKC inhibitor GF109203X completely abolished TPA-induced PLD activity, however, it only inhibited NMB-induced PLD activity by 20%. The combination of thapsigargin and GF109203X had the same effect as GF109203X alone. These data indicate that NMB receptor activation is coupled to both PLC and PLD. In contrast to a number of other phospholipase C-coupled receptors, NMB receptor stimulated changes in [Ca2+]i do not contribute to PLD activation. Both PKC-dependent and PKC-independent mechanisms are involved in the NMB-stimulated PLD activation with the PKC-independent pathway predominating., (Copyright 1998 Elsevier Science B.V.)

Recent studies show CCK stimulates tyrosine phosphorylation (TYR PHOSP) of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti-phosphotyrosine mAb (4G10), anti-p125FAK mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight > 60,000 including a broad M(r) 110-130,000 and M(r) 70-80,000. An increase in TYR PHOSP of both p125FAK and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1 +/- 1.9-fold increase for p125FAK and 3.6 +/- 0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125FAK at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8. CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 microM for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 microM CCK-JMV and with 3 microM CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125FAK and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins).

Recent studies suggest that in some tissues GRP receptor activation can both stimulate phospholipase C and the adenylate cyclase pathway and that activation of the latter pathway may be important in mediating some of its well-described growth effects. However, other studies suggest GRP-R may not be coupled to adenylate cyclase. To investigate this possibility, in the present study we determined the coupling of the GRP receptors to each pathway in mouse, rat, and guinea pig pancreatic acini and compared it to that in mouse Swiss 3T3 cells and human SCLC cells, all of which possess well-characterized GRP receptors. Moreover, we tested the effect of PKC activation on the ability of GRP-related peptides to increase cAMP accumulation in these tissues. Changes in cAMP levels were determined with or without IBMX present, with or without forskolin, or both to amplify small increases in cAMP. In mouse, rat and guinea pig pancreatic acini, murine Swiss 3T3 cells and human SCLC cells, GRP-related peptides caused a 600%, 500%, 250%, 300% and 60% increase, respectively, in [3H]IP with 1-3 nM causing a half-maximal effect. In murine Swiss 3T3 cells, IBMX, forskolin, and IBMX plus forskolin caused a 300%, 3500% and 10500% increase in cAMP, respectively. GRP-related peptides and VIP caused an additional 70% increase in cAMP with GRP causing a half-maximal (EC50) increase in cAMP at 2.1 +/- 0.5 nM, which was not significantly different from the EC50 of 3.1 +/- 0.9 nM for increasing [3H]IP in these cells. GRP-related peptides did not stimulate increases in cAMP in mouse, rat or guinea pig pancreatic acini or in SCLC cells either alone, with IBMX or forskolin or both. However, in pancreatic acini IBMX, forskolin or both increased cAMP 3 to 8-, 10 to 500-, and 100 to 1000-fold increase and the addition of VIP caused an additional 20-, 2-, and 3-fold increase in cAMP in the different species. In mouse pancreatic acini with TPA alone or IBMX plus TPA, neither bombesin nor GRP increased cAMP. Furthermore, in mouse pancreatic acini, neither TPA nor TPA plus IBMX altered basal or VIP-stimulated increases in cAMP. In mouse Swiss 3T3 cells TPA significantly increased cAMP stimulated by Bn, GRP or VIP. These results demonstrated that GRP receptor activation in normal tissues from three different species and a human tumoral cell line do not result in adenylate cyclase activation, whereas in Swiss 3T3 cells it causes such activation. The results suggest that the difference in coupling to adenylate cyclase is likely at least partially due to a difference in coupling to an adenylate cyclase subtype whose activation is regulated by PKC. Therefore, the possible growth effects mediated by this receptor in different embryonic or tumoral cells through activation of adenylate cyclase are not likely to be an important intracellular pathway for these effects in normal tissues.

In order to determine whether tachykinins alter the function of chief cells and to characterize the receptors mediating the effect, we investigated the abilities of various substance P (SP)-related peptides to inhibit the binding of 125I-Bolton-Hunter labeled substance P (125I-BH-SP) and their abilities to alter cell function in dispersed chief cells from guinea pig stomach. Binding of 125I-BH-SP was saturable, reversible, time- and temperature-dependent and was inhibited by several SP-related peptides with relative potencies of SP = physalaemin (IC50:0.19 nM) > SP methyl ester (SP-ME) (IC50:3.3 nM) > eledoisin (IC50:6.1 nM) > neurokinin A (NKA) (IC50: 65 nM) > neurokinin B (NKB) (IC50:80 nM). Analyses of these binding data demonstrated that chief cells possess a high and low affinity class of binding sites. Neither 125I-NKA nor [phenylalanyl-3,4,5-3H]senktide demonstrated saturable binding to chief cells. Acid stripping experiments demonstrated rapid ligand internalization with 55% of the bound radioligand internalized by 10 min. Phospholipase C activating agents (carbachol, CCK-8), adenylate cyclase activating agents (secretin, VIP), TPA and the calcium ionophore, A23187, all inhibited the binding of 125I-BH-SP and it was due to inhibition of ligand internalization with no change in surface bound parameters. SP (0.1 microM) stimulated pepsinogen secretion but was 4-times less efficacious than CCK-8 (10 nM) or carbachol (1 mM). 10 nM SP stimulated a rapid increase in cytoplasmic free calcium concentration ([Ca2+]i) followed by a sustained elevation lasting 2 min. Single cell spectroscopy demonstrated SP (10 pM to 1 microM) did not cause calcium oscillations. The NK1 receptor antagonist, CP96,345 specifically inhibited the SP-stimulated changes in [Ca2+]i and pepsinogen secretion. The relative potencies of SP-related peptides to stimulate pepsinogen secretion and [Ca2+]i demonstrated a close agreement with their abilities to inhibit the binding of 125I-BH-SP, and comparison of the dose-response curves suggests occupation of the low affinity sites mediate changes in biologic activity. In conclusion, the present study demonstrates that chief cells possess a NK1 subtype of tachykinin receptor, occupation of the low affinity sites of this receptor cause calcium mobilization and pepsinogen secretion, and that binding to this receptor is regulated by agents that activate phospholipase C, adenylate cyclase, protein kinase C and calcium mobilization.

We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

In the course of examining the actions of the cholecystokinin octapeptide (CCK-8) on pancreatic acini we found that CCK-8 can stimulate release of the large-molecular-weight cytoplasmic protein, lactate dehydrogenase (LDH) by as much as 6-fold. CCK-8-stimulated LDH release is mediated by a CCK-preferring receptor, detectable at 100 pM CCK-8, maximal at 100 nM CCK-8, constant for up to 30 min, reversible, not desensitized, and dependent on oxidative metabolism and incubation temperature but not on calcium in the extracellular medium. This action of CCK-8 is blocked by inhibitors of protein kinases, staurosporine, H-7, H-8 and H-9, but not by calmodulin antagonists, chlorpromazine, trifluoperazine or W-7. This action of CCK-8 on LDH release is not reproduced by TPA, 8Br-cAMP or A23187. Thus, it appears to be mediated by a previously uncharacterized protein kinase or an isoform of protein kinase C that is not maximally stimulated by TPA.

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Recent cloning studies confirm two subtypes of Bn receptors exist, a neuromedin B-preferring receptor (NMB-R) and a gastrin-releasing peptide-preferring receptor (GRP-R). Both subtypes occur widely in GI tract and the CNS; however, in contrast to the GRP-R subtype little is known about the ligand-receptor interactions for the NMB-R. Therefore, in the present study we explored the ligand-receptor interactions including kinetics, stoichiometry, internalization, degradation and regulation by guanine nucleotide binding proteins with the NMB-R and compared it to the GRP-R. The rat glioblastoma C-6 cell line which possess functional NMB-R and 3T3 cells which possess functional GRP-R were used. 125I-[D-Tyr0]NMB and 125I-[Tyr4]Bn were prepared using Iodogen and purified on HPLC. At 37 degrees C binding of 125I-[D-Tyr0]NMB to NMB-R or 125I-[Tyr4]Bn to GRP-R was maximal by 5-15 min and decreased to 60-70% after 60 min. HPLC analysis of the 60 min supernatant showed that > 80% of each tracer was degraded. Addition of proteinase inhibitors had a varied inhibitory effect on degradation with the relative order of potency in C-6 cells being leupeptin > bacitracin < chymostatin > phosphoramidon >> bestatin and amastatin and 3T3 cells being bacitracin = phosphoramidon > leupeptin = bestatin > chymostatin > amastatin in 3T3 cells. By HPLC analysis addition of bacitracin prevented the degradation in both cell types. With both receptor subtypes dissociation of bound radioligands was slow, with 70-80% of either 125I-[D-Tyr0]NMB or 125I-[Tyr4]Bn remained cell-associated after 60 min suggesting possible peptide internalization. With an acid wash procedure to remove surface bound radioligands, 60% of the C-6 cell-associated 125I-[D-Tyr0]NMB and 52% of the 3T3 cell-associated 125I-[Tyr4]Bn were internalized after 30 min at 37 degrees C. With membranes from cells possessing either receptor subtype, the stable guanine nucleotide GPP(NH)P inhibited in a dose-dependent fashion binding of ligands. Computer analysis demonstrated that GPP(NH)P decreased receptor affinity for ligands to both receptor subtypes. These results demonstrated that NMB receptors, similar to GRP receptors and rapidly internalize bound agonists and rapidly degrade agonists. The ligand-receptor interaction is regulated by a guanine nucleotide binding protein for both Bn receptor subtypes.

In dispersed gastric chief cells from guinea-pig stomach, binding of iodinated cholecystokinin octapeptide (125I-CCK-8) was relatively slow, temperature-dependent, to a single class of binding sites and inhibited by various gastrin- and CCK-related agonists and receptor antagonists. Binding of iodinated gastrin-I (125I-gastrin-I) was moderately rapid, temperature-dependent, to a single class of binding sites, and inhibited by various gastrin and CCK-related agonists and receptor antagonists. Gastrin-I as well as C-terminal fragments of CCK containing from eight amino acids (CCK-8) to four amino acids (CCK-4) stimulated pepsinogen secretion and inhibited binding of 125I-CCK-8 and 125I-gastrin-I. In addition, each of five different receptor antagonists inhibited binding of 125I-CCK-8 and 125I-gastrin-I and inhibited pepsinogen secretion stimulated by CCK-8 or gastrin-I. With each of eight different agonists and with each of five different antagonists the value of IC50 for inhibition of binding of 125I-CCK-8 was not significantly different from the value of IC50 for inhibition of binding of 125I-gastrin-I, indicating that in gastric chief cells the sites to which 125I-CCK-8 binds are the same sites to which 125I-gastrin-I binds. With the agonists as well as with the antagonists, however, there was no consistent relationship between the ability of a particular agent to inhibit binding and its ability to modify pepsinogen secretion, indicating that in gastric chief cells the sites that bind 125I-CCK-8 and 125I-gastrin-I are not the receptors that mediate stimulation of pepsinogen secretion by CCK-8 or by gastrin-I.

COOH-terminal fragments of cholecystokinin varying in length from 1 to 3 amino acids and their NH2-terminal butyloxycarbonyl derivatives were investigated for their ability to interact with the cholecystokinin receptor on dispersed acini from guinea pig pancreas. No fragment stimulated amylase secretion when present alone, but each of the butyloxycarbonyl derivatives and the COOH-terminal tripeptide amide inhibited the stimulation of enzyme secretion by cholecystokinin. In each case the inhibition was surmounted by increasing the concentration of cholecystokinin. Each fragment also inhibited binding of 125I-labeled cholecystokinin, with significant inhibition occurring with 30 microM butyloxycarbonyl tripeptide amide, 0.3 mM butyloxycarbonyl dipeptide amide, 10 mM butyloxycarbonyl phenylalanine amide and 3 mM tripeptide amide of cholecystokinin. In each case, there was a close correlation between the ability of the fragment to inhibit binding of 125I-labeled cholecystokinin and its ability to inhibit cholecystokinin-stimulated amylase release, cholecystokinin-stimulated 45Ca outflux and cholecystokinin-stimulated residual stimulation of amylase secretion. The inhibition of amylase secretion caused by the butyloxycarbonyl tripeptide of cholecystokinin was reversible and specific for those peptides which interact with the cholecystokinin receptor (i.e., cholecystokinin, caerulein, gastrin); it did not inhibit the actions of bombesin, carbachol, physalaemin, vasoactive intestinal peptide, secretin, PHI, ionophore A23187 or 8-bromo cyclic AMP. These results demonstrate that COOH-terminal fragments of cholecystokinin comprise a new class of cholecystokinin receptor antagonists.

None of six different tryptophan-modified analogues of the C-terminal octapeptide of cholecystokinin differed from the unaltered peptide in terms of their efficacies for stimulating amylase secretion from dispersed acini prepared from guinea-pig pancreas. Replacement of hydrogen with fluorine in position 5 or 6 on the indole ring of the tryptophan residue did not alter the potency with which the peptide stimulated amylase secretion; however, replacement of hydrogen by fluorine in positions 4, 5, 6 and 7 of the indole ring, or modifying or replacing the indole nitrogen caused a 30- to 300-fold decrease in potency. Changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin. Our findings indicate that reducing the ability of the tryptophan residue to donate electrons produced a greater decrease in the affinity of the peptide for cholecystokinin receptors than did abolishing the ability of tryptophan to form hydrogen bonds, and modification that altered both abilities caused a greater decrease in affinity than did modification of only one ability. Finally, in the tryptophan residues of cholecystokinin octapeptide, tetrafluorination of the indole ring or replacing the indole nitrogen by oxygen reduced the ability of the peptide to cause residual stimulation of enzyme secretion, probably by accelerating the rate at which bound peptide dissociated from its receptors when the acini were washed and resuspended in fresh incubation solution.

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3':5' monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 microM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1-2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.

When pancreatic acini are incubated with increasing concentrations of cholecystokinin octapeptide (CCK-8) the dose-response curve for stimulation of enzyme secretion increases, reaches a maximum and then decreases. The upstroke of the dose-response curve reflects occupation of high-affinity, stimulatory CCK receptors (Kd 69 pM), whereas the downstroke of the dose-response curve reflects occupation of low-affinity inhibitory CCK receptors (Kd 10 nM). In the present study, we used dispersed acini from rat pancreas to examine the effects of CCK-JMV-180, an analogue of the C-terminal heptapeptide of CCK (CCK-7) having the structure BOC-Tyr(SO3) Ahx-Gly-Trp-Ahx-Asp2 phenylethyl ester. CCK-JMV-180 inhibited binding of 125I-labelled CCK-8 to pancreatic acini. Computer analysis of the dose-inhibition curve indicated that CCK-JMV-180 interacted with both classes of CCK receptor and had a Kd of 2.2 nM for high-affinity CCK receptors and a Kd of 19 nM for low-affinity CCK receptors. Occupation of high-affinity CCK receptors by CCK-JMV-180 caused a 14-fold increase in amylase secretion, the same increase caused by occupation of these high-affinity receptors by CCK-7 or CCK-8. Occupation of low-affinity CCK receptors by CCK-JMV-180 did not alter amylase secretion, in contrast to occupation of these low-affinity receptors by CCK-7 or CCK-8, each of which caused inhibition of amylase secretion. Furthermore, occupation of the low-affinity CCK receptors by CCK-JMV-180 reversed the inhibition of amylase secretion caused by a supramaximal concentration of CCK-8. The present results indicate that CCK-JMV-180 interacts with high-affinity CCK receptors and with low-affinity CCK receptors, and has a functionally distinct action at each class of receptor: CCK-JMV-180 is an agonist at the high-affinity receptors and a competitive antagonist at the low-affinity receptors.

When dispersed acini from mouse pancreas are first incubated with cholecystokinin octapeptide, washed and then reincubated with no additions there is significant stimulation of amylase secretion during the second incubation (residual stimulation of enzyme secretion). Cholecystokinin-induced residual stimulation of enzyme secretion is modified, but not abolished, by reducing the temperature of the first incubation from 37 degrees C to 4 degrees C. Measurement of binding of 125I-labeled cholecystokinin octapeptide indicated that maximal cholecystokinin induced residual stimulation of enzyme secretion occurs when 12-20% of cholecystokinin receptors are occupied by cholecystokinin octapeptide. Moreover, maximal cholecystokinin-induced residual stimulation of amylase secretion is 25% greater than maximal cholecystokinin-induced direct stimulation of amylase secretion. Cholecystokinin tetrapeptide, which causes the same maximal direct stimulation of amylase secretion as does cholecystokinin octapeptide, causes a maximal residual stimulation of enzyme secretion that is only 30% of that caused by a maximally effective concentration of cholecystokinin octapeptide. Adding dibutyryl cyclic GMP to the second incubation can reverse the residual stimulation caused by adding cholecystokinin to the first incubation. The pattern and extent of the dibutyryl cyclic GMP-induced reversal of residual stimulation varies, depending on the temperature and concentration of cholecystokinin octapeptide in the first incubation. The present results are compatible with the hypothesis that mouse pancreatic acini possess two classes of cholecystokinin receptors. One class has a relatively high affinity for cholecystokinin and produces stimulation of enzyme secretion; the other class has a relatively low affinity for cholecystokinin and produces inhibition of enzyme secretion.

Benzotript, N-p-chlorobenzoyl-L-tryptophan, is a specific cholecystokinin receptor antagonist. In the present study we used dispersed pancreatic acini to examine tryptophan as well as several different N-acyl derivatives of tryptophan for their abilities to function as cholecystokinin receptor antagonists. L-Tryptophan, D-tryptophan as well as each acyl derivative tested inhibited cholecystokinin-stimulated amylase secretion and outflux of 45Ca and there was a good correlation between the ability of a particular agent to inhibit the action of cholecystokinin on acinar function and its ability to inhibit binding of 125I-labeled cholecystokinin to pancreatic acini. Results with butyloxycarbonyl-L-tryptophan indicated that the inhibition of the action of cholecystokinin caused by L-tryptophan and various acyl derivatives is specific, competitive and fully reversible. In functioning as a cholecystokinin receptor antagonist the relative potencies of the agents tested were: carbobenzoxy-L-tryptophan greater than benzotript greater than benzoyl-L-tryptophan = butyloxycarbonyl-L-tryptophan greater than acetyl-L-tryptophan greater than L-tryptophan. In inhibiting the actions of cholecystokinin, native as well as N-acyl derivatives of D-tryptophan were equipotent with the corresponding compound containing L-tryptophan. Although L-tryptophan inhibited the actions of cholecystokinin, L-phenylalanine, L-methionine or L-aspartic acid, even when tested at concentrations as high as 3 mM, did not alter the action of cholecystokinin on pancreatic acini. The antagonism of the actions of cholecystokinin was not restricted to N-acyl derivatives of L-tryptophan because butyloxycarbonyl-L-methionine and butyloxycarbonyl-L-phenylalanine but not butyloxycarbonyl-L-aspartic acid also antagonized the actions of cholecystokinin. These results demonstrate that both the nature of the N-acyl group and the amino acid residue are important determinants of the affinity of the antagonist for the cholecystokinin receptor. For derivatives of L-tryptophan, the more hydrophobic the N-acyl moiety, the greater the affinity of the derivative for the cholecystokinin receptor.

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by beta-alanine caused a 300-fold decrease in the potency with which the peptide stimulated enzyme secretions, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The beta-alanine-substituted peptide was approximately ten times more potent when the n terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin. The magnitude of stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same with each analogue as it was with the unaltered peptide. Replacing the aspartyl residue by beta-alanine or glutamic acid or replacing the N-terminal t-butyloxycarbonyl moiety by benzyloxycarbonyl caused an equivalent decrease in the ability of the peptide to stimulate enzyme secretion and its ability to cause residual stimulation of enzyme secretion. In contrast, the N-terminal desamino analogue of cholecystokinin heptapeptide was ten times less potent than the unaltered peptide in stimulating amylase secretion, but 100 times less potent than the unaltered peptide in causing residual stimulation of enzyme secretion.