Effect of phenylephrine and prazosin on the somatostatinergic system in the rat frontoparietal cortex

Somatostatin (SS) and noradrenaline (NA) are distributed in the rat cerebral cortex, and seizure activity is one of the aspects of behavior affected by both neurotransmitters. Due to the possible interaction between both neurotransmitter systems, we studied whether phenylphrine, an ₁-adrenoceptor agonist, and prazosin, an ₁-adrenoceptor antagonist, can modulate SS-like immunoreactivity (SS-LI) levels, binding of [¹²⁵I][Tyr¹¹]SS to its specific receptors, the ability of SS to inhibit adenylate cyclase (AC) activity, and the guanine nucleotide binding regulatory protein G_i and G_o in the Sprague-Dawley rat frontoparietal cortex. An IP dose of 2 or 4 mg/kg of phenylephrine injected 7 h before decapitation decreased the number of SS receptors and increased the apparent affinity in frontoparietal cortex membranes. An IP dose of 20 or 25 mg/kg of prazosin administered 8 h before decapitation increased the number of SS receptors and decreased their apparent affinity. The administration of prazosin before the phenylephrine injection prevented the phenylephrine-induced changes in SS binding. The addition of phenylephrine and/or prazosin 10⁻⁵ M to the incubation medium changed neither the number nor the affinity of the SS receptors in the frontoparietal cortex membranes. Phenylephrine or prazosin affected neither SS-LI content nor the basal or forskolin (FK)-stimulated AC activities in the frontoparietal cortex. In addition, SS caused an equal inhibition of AC activity in frontoparietal cortex membranes of phenylephrine- and prazosin-treated rats compared with the respective control group. Finally, phenylephrine and prazosin did not vary the pertussis toxin (PTX)-catalyzed ADP ribosylation of G_i- and/or G_o-proteins. These results suggest that the above-mentioned changes are related to the phenylephrine activation of ₁-adrenoceptors or to the blocking of these receptors by prazosin. In addition, these data provide further support for a functional interrelationship between the ₁-adrenergic and somatostatinergic systems in the rat frontoparietal cortex.     

Effect of nitric oxide on the somatostatinergic system in the rat exocrine pancreas

Nitric oxide (NO) and somatostatin (SS) are two important mediators of the exocrine and endocrine pancreas, exerting opposite effects on this organ. There is strong evidence suggesting an interaction between pancreatic NO and SS. The aim of this study was to determine whether L-arginine (L-Arg), the substrate for NO synthase (NOS), and Nomega-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, regulate pancreatic somatostatin-like immunoreactivity (SSLI) content and the SS mechanism of action in pancreatic acinar cell membranes. L-Arg (150 mg/kg, intraperitoneally (i.p.)), L-NAME (50 mg/kg, i.p.) or L-NAME plus L-Arg were injected twice daily at 8 h intervals for 8 days. L-Arg decreased pancreatic SSLI content as well as the number of SS receptors in pancreatic acinar cell membranes whereas L-NAME increased both parameters. The stable SS analogue SMS 201-995 induced a significantly lower inhibition of forskolin-stimulated adenylyl cyclase activity in pancreatic acinar cell membranes from L-Arg-treated rats whereas an increased inhibition was observed in pancreatic acinar membranes from L-NAME-treated rats. These results indicate that the NO system may contribute to the regulation of the pancreatic somatostatinergic system.     

Gastrointestinal growth factors and hormones have divergent effects on Akt activation

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.     

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.     

Hormonal control of ADP-ribosyl cyclase activity in pancreatic acinar cells from rats

Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the beta-NAD+ analogs 1,N6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.     

The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules

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.     

The effect of concomitant stimulation with cholecystokinin and epidermal growth factor on extracellular signal-regulated kinase (ERK) activity in pancreatic acinar cells.

The transmission of extracellular proliferation and differentiation signals into their intracellular targets is mediated by a signaling cascade culminating in mitogen-activated protein kinase (MAPK) also known as ERK. In pancreatic acinar cells both cholecystokinin (CCK) and epidermal growth factor (EGF) are known to stimulate ERK. Regulatory interactions among individual receptor-coupled signaling cascades are critically important for establishing cellular responses in the face of multiple stimuli. The aim of our study was to evaluate the effect of concomitant stimulation of G protein-coupled receptors (GPCR) and EGF receptors on ERK activity in isolated pancreatic acinar cells. ERK activity was determined by means of Western-blotting, with the use of the antibody which recognizes active, tyrosine-phosphorylated kinase (pY-ERK). pY-ERK level was strongly elevated by 10 nM CCK-8, 100 microM carbachol (CAR), or 100 nM EGF. The addition of EGF to 60 min-lasting incubations of acini with CCK-8 or CAR caused abrupt decrease of pY-ERK level to 56 and 59% of control, respectively. Similar phenomenon was observed when short stimulation with CCK-8 or CAR was superimposed on the effect of EGF. After the addition of EGF to acini incubated previously with phorbol ester TPA, strong decrease in pY-ERK level was also observed. In conclusion, in pancreatic acinar cells, concomitant stimulation with CCK or CAR and EGF has strong inhibitory effect on ERK cascade. This inhibitory cross-talk may be mediated, at least partially, by protein kinase C (PKC). These mutual inhibitory interactions demonstrate novel mechanism for integration of multiple signals generated by activation of G-protein-coupled and growth factor receptors in pancreatic acinar cells.     

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca(2+)-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca(2+)](i) decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK(A) receptor states. CCK stimulated an association of Lyn with PKC-delta, Shc, p125(FAK) and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130(CAS) or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125(FAK), PYK2, PKC-delta and Shc, which play central roles in CCK's effects on acinar cell function.     

The activation of neuronal NO synthase is mediated by G-protein betagamma subunit and the tyrosine phosphatase SHP-2.

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L-NAME and alpha-guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK-induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP-2 mutant and the betaARK1 (495-689) sequestrant peptide, indicating the requirement of activated SHP-2 and G-betagamma subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP-2 activity) revealed that in response to CCK-treatment, SHP-2 associated to G-beta1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.     

Calcineurin mediates pancreatic growth in protease inhibitor-treated mice

CCK acts on pancreatic acinar cells to increase intracellular Ca(2+) leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca(2+) and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.     

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, Gi function, and somatostatin receptors in rat exocrine pancreas.

To analyze the effect of bombesin on the somatostatin (SS) mechanism of action in the exocrine pancreas, male Wistar rats (250-270 g) were injected intraperitoneally with bombesin (10 microg/kg) three times daily at 8-h intervals for 7 or 14 days. Bombesin attenuated the ability of SS to inhibit forskolin-stimulated adenylyl cyclase activity in pancreatic acinar membranes. However, it did not decrease the ability of forskolin to stimulate the adenylyl cyclase catalytic subunit. The ability of 5'-guanylylimidodiphosphate [Gpp(NH)p] (a nonhydrolyzable GTP analog) to inhibit forskolin-stimulated adenylyl cyclase activity was diminished in pancreatic acinar cell membranes from bombesin-treated rats. Bombesin administration did not affect the ADP-ribosylation of a 41-kDa G protein catalyzed by pertussis toxin. The maximal SS binding capacity of pancreatic acinar membranes from bombesin-treated rats was decreased when compared with controls at the two time periods studied. The bombesin/gastrin-releasing peptide antagonist [D-Tpi6,Leu13psi(CH2NH)Leu14]bombesin (6-14) (RC-3095) (10 microg/kg i.p.), injected three times daily at 8-h intervals for 7 or 14 days, had a similar effect to that of bombesin on the SS mechanism of action. The combined administration of bombesin and its antagonist RC-3095 had a greater effect on the SS receptor-effector system than when administered separately. The present study indicates that the pancreatic SS receptor-effector system may be regulated by bombesin in vivo.     

Effect of ligands that increase cAMP on caerulein-induced zymogen activation in pancreatic acini

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 muM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.     

Guanylate cyclase, a cell surface receptor

Guanylate cyclase appears to represent a central member of a diverse family of proteins involved in cell signaling mechanisms including the protein kinases, a low Mr ANP receptor, and possibly adenylate cyclase (based on limited sequence identity with the yeast enzyme). A membrane form of guanylate cyclase represents a new model for cell surface receptors, although such a model was once envisioned for adenylate cyclase (79). In original models for adenylate cyclase, hormone was thought to bind with either the enzyme or with an unknown protein to enhance cyclic AMP production (79). Guanylate cyclase appears to fall into the first adenylate cyclase model where binding of a ligand to an extracellular site on the enzyme transmits a signal to an intracellular catalytic site. The production of cyclic GMP, a second messenger, and of pyrophosphate are then increased. The protein tyrosine kinase family of receptors (80) and possibly another forthcoming family of cell surface receptors containing protein tyrosine phosphatase activity (81-83) contain a single transmembrane domain like guanylate cyclase. Furthermore, the protein tyrosine kinases are activated by ligand binding to the extracellular domain. However, the activation of guanylate cyclase, unlike these cell surface receptors, results in the formation of a low molecular weight second messenger.     

Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

Effect of proglumide, a cholecystokinin receptor antagonist, on caerulein-stimulated pancreatic enzyme secretion and pancreatic polypeptide release in the dog

In five conscious dogs we studied the effect of proglumide, a cholecystokinin (CCK) antagonist, on caerulein-stimulated pancreatic secretion and release of pancreatic polypeptide (PP). Graded doses of caerulein (15-240 ng/kg per h) were infused intravenously. Experiments were repeated with a fixed infusion of proglumide (40 mg/kg per h). Release of PP following increasing doses of caerulein was significantly inhibited by proglumide (P less than 0.01). However, proglumide did not significantly affect caerulein-stimulated pancreatic protein secretion. Proglumide might be useful in defining the physiological role of CCK.     

Cholecystokinin-stimulated protein kinase C-delta kinase activation,tyrosine phosphorylation,and translocation are mediated by Src tyrosine kinases in pancreatic acinar cells

Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.     

Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells

The effects of activating the G_qprotein-coupled cholecystokinin (CCK) receptor on differentproteins/signaling molecules in the mitogen-activated protein kinase(MAPK) cascade in pancreatic acinar cells were analyzed and comparedwith the effects of activating the tyrosine kinase-coupled epidermalgrowth factor (EGF) receptor. Both EGF and CCK octapeptide rapidlyincreased the activity of the MAPKs [extracellular signal-regulatedkinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-inducedincrease of MAPK activity was transient, with only a slight elevationafter 30 min, whereas CCK-stimulated MAPK remained at a high level ofactivation to 60 min. The protein kinase C inhibitor GF-109203Xabolished the activation by phorbol ester and inhibited the effect ofCCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a muchlarger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereasEGF activated both MEKs by only 2-fold. Immunoblotting revealed threedifferent forms of Raf in pancreatic acinar cells. Of the total basalRaf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% wasc-Raf-1. All three forms of Raf were stimulated to a greater extent byCCK than by EGF, which was especially evident for Raf-A and c-Raf-1.The effect of CCK in activating Rafs was at least partially mimicked bystimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantlyincreased GTP-bound Ras by 183 and 164% at 2.5 and 10 min,respectively; CCK and TPA had no measurable effect. Our study suggeststhat CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.

     

Diurnal pattern of rat pancreatic acinar cell replication

Fully differentiated pancreatic acinar cells can enter the cell cycle under appropriate conditions in the rat. The aim of this study was to analyse the diurnal pattern of acinar cell proliferation as a function of food intake and the release of cholecystokinin (CCK), because the peptide hormone CCK is a major physiological regulator of rat pancreatic acinar cell replication. Pancreatic acinar cell replication was quantitated using an antibody against the S-phase marker proliferating cell nuclear antigen (PCNA). In addition, acinar cells in S-phase were detected after injecting bromodeoxyuridine (BrdU) and subsequent immunohistochemical staining of BrdU-positive nuclei. Rat pancreata were analysed during the day under standard diet conditions, as well as after various schedules of fasting and refeeding and after the application of the CCK receptor antagonist L-364,718. Between 6 a.m. and 6 p.m., the PCNA labeling index was 4.4±0.9%, while between 9 p.m. and 3 a.m. the PCNA labeling index was elevated and reached peak values of 11.4% (mean value: 7.8±2.5%) around midnight. BrdU-positive cells also doubled around midnight, compared to the 9:00 a.m. value. In fasted rats, acinar cell proliferation was completely suppressed and this suppression could be overcome by injection of the CCK analog cerulein. In addition, the CCK antagonist L-364,718 led to the same results as fasting. Here we show for the first time that there is a diurnal pattern of pancreatic acinar cell proliferation in rats, which is dependent on food intake and is mediated by CCK.     

Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist

Partial agonists are effective tools for advancing developmentof highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of apartial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in nativepancreatic acinar cells and Chinese hamster ovary cells expressing CCKreceptor (CHO-CCKR). We developed and characterized a novel fluorescentpartial agonist,rhodamine-Gly-[(Nle^28,31)CCK-26-32]-phenethylester, that binds specifically and with high affinity to CCK receptors.Such analogs are fully efficacious pancreatic acinar cell secretagogueswithout supramaximal inhibition that mobilize intracellular calciumwith little or no increase in phospholipase C (PLC) activity. Despiteminimal phosphorylation of CCK receptors in response to this partialagonist, receptor trafficking was the same as that observed with fullagonist (CCK). This included normal internalization viaclathrin-dependent endocytosis in CHO-CCKR cells and insulation on thesurface of pancreatic acinar cells. Also, as with CCK-occupiedreceptor, fluorescence recovery after photobleaching of partialagonist-occupied receptor on the acinar cell surface demonstrated amarked temperature-dependent slowing of its rate of diffusion. This wassimilarly associated with resistance to acid-induced dissociation ofligand. Thus some key molecular regulatory mechanisms for CCK receptorinternalization and insulation may be initiated by cellular signalingcascades that are not dependent on PLC activation or receptor phosphorylation.     

Low affinity cholecystokinin receptor inhibits cholecystokinin- and bombesin-induced oscillations of cytosolic Ca2+ concentration

We have investigated whether low affinity cholecystokinin (CCK) receptors suppress agonist-induced rises of cytosolic free Ca(2+) concentration ([Ca(2+)]c) in pancreatic acinar cells by using properties of caffeine. A high concentration of caffeine (20 mM) completely blocked inositol 1,4,5-trisphosphate (InsP(3))-induced [Ca(2+)]c rises but spared the InsP(3)-independent long-lasting [Ca(2+)]c oscillations. In the presence of 20 mM caffeine, only high concentrations of CCK, but not bombesin or JMV-180, suppressed the caffeine-resistant CCK or bombesin-induced [Ca(2+)]c oscillations, indicating that low affinity CCK receptors inhibit agonist-induced [Ca(2+)]c oscillations. It could be one of the underlying mechanisms by which low affinity CCK receptors suppress secretion in pancreatic acinar cells.