The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.     

Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.     

Phorbol ester inhibits cholecystokinin octapeptide-induced amylase secretion and calcium mobilization, but is without effect on secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in rabbit pancreatic acini

The effect of prolonged protein kinase C activation on cholecystokinin octapeptide (CCK-8)-induced amylase secretion from rabbit pancreatic acini was studied by means of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). The phorbol ester itself increased basal amylase secretion but inhibited completely the secretory response to relatively low concentrations of CCK-8. The inhibitory action of TPA on CCK-8-induced amylase secretion was paralleled by inhibition of CCK-8-induced calcium mobilization but not by inhibition of CCK-8-induced breakdown of 32P-labelled phosphatidylinositol 4,5-bisphosphate. The results presented suggest that protein kinase C, or one of its phosphorylated products, inhibits the CCK-8-stimulated pathway leading to secretion at a level beyond the secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inhibition of the initial, inositol 1,4,5-trisphosphate-mediated and extracellular calcium-independent, increase in free cytosolic calcium concentration, together with the findings of others, suggests that the efficacy of this inositol-phosphate to release calcium is reduced.     

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.     

ATP-consuming and ATP-generating enzymes secreted by pancreas

Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid chromatography analysis additionally shows that this enzyme has broad substrate specificity toward other nucleotides, UTP, UDP, ITP, and IDP. In addition, secretion contains ecto-5'-nucleotidase, CD73, further converting [(3)H]AMP to adenosine. Along with highly active hydrolytic enzymes, there were also ATP-generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes release of both ATP-consuming and ATP-generating enzymes into pancreatic juice. This newly discovered richness of secreted enzymes underscores the importance of purine signaling between acini and pancreatic ducts lumen and implies regulation of the purine-converting enzymes release.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

PGE2 regulates cholecystokinin-octapeptide (CCK-8)-stimulated Cl- conductance in isolated zymogen granules from rat pancreas.

In this study we have examined the effects of prostaglandin E2 (PGE2), the cyclooxygenase inhibitor, indomethacin, and a protein kinase A inhibitor (PKA-I) on the Cl- conductance in isolated zymogen granules (ZG) from cholecystokinin octapeptide (CCK-8) pre-stimulated pancreatic acini. The Cl- conductance in isolated ZG from CCK-8 pre-stimulated rat pancreatic acini increases with increasing CCK-8 concentrations and decreases at supramaximal CCK-8 concentrations. The basal and CCK-8-stimulated Cl- conductance in ZG is inhibited by pretreatment of acini with PGE2 (10(-6) M). This PGE2-induced inhibition is abolished in the presence of PKA-I (20 U/ml). Furthermore, pretreatment of acini with indomethacin (10(-5) M) or PKA-I (20 U/ml) abolishes the decrease in the CL- conductance at supramaximal CCK-8 concentrations (10(-9) M). We conclude that the inhibition of the CL- conductance in isolated ZG at high CCK-8 concentrations is mediated by an enhanced production of PGE2, and that PGE2 operates by stimulating adenylate cyclase (AC) with a consequent rise in cAMP and activation of PKA.     

Exogenous leptin inhibits the secretion of pancreatic juice via a duodenal CCK1-vagal-dependent mechanism in anaesthetized rats

Leptin originally described as product of the ob gene has been shown to be expressed in various tissues including the gastrointestinal tract. In this study, we investigated the influence of leptin on the secretion of pancreatic juice in biliary-pancreatic duct cannulated anaesthetised rats and in dispersed rat pancreatic acini in vitro. Exogenous leptin was given in boluses intravenously with or without CCK-8 (12 pmol kg(-1) body weight) in the presence or absence pharmacological CCK(1) receptor blockade, cervical vagotomy, and capsaicin pre-treatment. Administration of leptin (0.1, 1 and 10 microg kg(-1) body weight) did not affect the volume of bile and pancreatic juice while the protein and trypsin outputs were reduced in a dose-dependent manner. In the rats, leptin inhibited CCK-8 stimulated protein and trypsin outputs stronger than the basal pancreatic secretion. The inhibition by leptin was abolished by the pharmacological CCK(1) receptor blockade, cervical vagotomy, and capsaicin pre-treatment. In contrast, leptin did not affect basal and CCK-8-stimulated amylase release from the dispersed rat pancreatic acini in vitro. In conclusion, the results of the present study suggest that leptin does not act directly on the rat pancreatic acinar cells but inhibits the secretion of pancreatic enzymes acting indirectly via a neurohormonal CCK-vagal-dependent mechanism.     

Ontogeny of secretory function and cholecystokinin binding capacity in immature rat pancreas

Isolated acini were prepared from the pancreas of immature rats (age less than 1 hr. - 48 hrs) in order to study the development of the secretory process. The ultrastructural integrity of the acinar cells was maintained after digestion and stimulation with secretagogues. Acini prepared from rats aged 24 - 48 hours responded to both CCK-8 and carbachol with significant increases in amylase release. Although typical biphasic dose response curves were obtained, the curves were shifted to the right by 1 - 2 log units, compared to the responses of adult acini. At ages younger than 24 hours, acini were insensitive to secretagogues but were sensitive to the calcium ionophore A23187. CCK receptors were virtually absent from membranes prepared from newborn pancreases, but binding of CCK, although small, was measurable at 12 hours and slowly increased up to 48 hours. A greater amount of binding was seen at 72 hours, which appeared constant up to 14 days. At 21 days, adult levels of binding were found. These results confirm previous studies that the rat pancreas is insensitive to secretagogues in the first 24 hours of life. After age 24 hours the secretory process is intact but less sensitive to secretory agents than the more mature pancreas. In the case of CCK, this may be due to lesser numbers of CCK receptors and/or affinity of CCK for its receptor.     

Gastrointestinal hormones cause rapid c-Met receptor down-regulation by a novel mechanism involving clathrin-mediated endocytosis and a lysosome-dependent mechanism

The activated c-Met receptor has potent effects on normal tissues and tumors. c-Met levels are regulated by hepatocyte growth factor (HGF); however, it is unknown if they can be regulated by gastrointestinal (GI) hormones. c-Met is found in many GI tissues/tumors that possess GI hormone receptors. We studied the effect of GI hormones on c-Met in rat pancreatic acini, which possess both receptors. CCK-8, carbachol, and bombesin, but not VIP/secretin, decreased c-Met. CCK-8 caused rapid and potent c-Met down-regulation and abolished HGF-induced c-Met and Gab1 tyrosine phosphorylation, while stimulating c-Met serine phosphorylation. The effect of cholecystokinin (CCK) was also seen in intact acini using immunofluorescence, in a biotinylated fraction representing membrane proteins, in single acinar cells, in Panc-1 tumor cells, and in vivo in rats injected with CCK. CCK-8 did not decrease cell viability or overall responsiveness. GF109203X, thapsigargin, or their combination partially reversed the effect of CCK-8. In contrast to HGF-induced c-Met down-regulation, the effect of CCK was decreased by a lysosome inhibitor (concanamycin) but not the proteasome inhibitor lactacystin. Inhibitors of clathrin-mediated endocytosis blocked the effect of CCK. HGF but not CCK-8 caused c-Met ubiquitination. These results show CCK and other GI hormones can cause rapid c-Met down-regulation, which occurs by a novel mechanism. These results could be important for c-Met regulation in normal as well as in neoplastic tissue in the GI tract.     

Evaluation of the role of calcium in cytotoxic injury in isolated rat pancreatic acini

The role of extracellular Ca2+ in pancreatic acinar membrane damage (cellular injury) by nicotine, membrane-active agents (mellitin, snake venom and Ca2+ ionophore A23187) and secretagogues (CCK-8 and secretin) was investigated. Freshly isolated dispersed pancreatic acini from 18 h fasted adult rats were incubated with one of the aforementioned agents, in the absence and presence of Ca2+. Cellular injury was assessed by measuring the release of pulse-labeled 51Cr and LDH. In addition, release of amylase, trypsinogen and chymotrypsinogen was also determined. In the absence of Ca2+ nicotine (6 mM) caused a profound release of 51Cr and LDH as well as amylase, trypsinogen and chymotrypsinogen from the isolated pancreatic acini. Release of these enzymes and 51Cr decreased sharply with addition of increasing concentrations (0.25-5 mM) of Ca2+. Release of 51Cr and amylase by snake venom (50 micrograms/ml) was found to be 100 and 25% higher, respectively, in the absence of Ca2+ than in its presence. On the other hand, the Ca2+ ionophore A23187 (7 micrograms/ml) was found to be effective in releasing 51Cr and amylase only in the presence of Ca2+. CCK-8, (0.25nM), secretin (1 microM) and mellitin (0.5 microgram/ml) although significantly stimulated amylase secretion (225-350%) in the presence of Ca2+, none of the agents induced 51Cr release from acini, either in the absence or in the presence of extracellular Ca2+. It is concluded that the extracellular Ca2+ plays no specific role in cytotoxic injury in isolated pancreatic acini.     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Cholecystokinin octapeptide induces both proliferation and apoptosis in the rat pancreas

Cholecystokinin-8 (CCK-8) causes exocrine pancreatic hypertrophy and hyperplasia. High doses of the CCK analogue cerulein causes necrosis and an inflammatory response in the pancreas. We have studied the pancreatic growth response in rats after administration of CCK-8 for 3 days, given either intermittently (20-80 microg/kg) twice a day, or continuously (2.4-48 microg/kg per 24 h). Plasma CCK-8 levels, pancreatic wet weight, water, protein and DNA contents and the pancreatic caspase-3 activity were measured. Cell proliferation was visualized by [3H]thymidine incorporation and apoptosis by TUNEL reaction. Continuous administration of CCK-8 dose-dependently increased the plasma CCK levels, the pancreatic wet weight, protein and DNA contents as well as thymidine labeling index, apoptotic index and caspase-3 activity. Intermittent injections of CCK-8 caused transient raises in plasma CCK, increased apoptotic index and caspase-3 activity, a dose-dependent increase in thymidine labeling but caused a dose-dependent reduction of pancreatic wet weight, protein, and DNA contents. It is concluded that CCK-8 causes both increased proliferation and apoptosis in the pancreas. In case of continuous administration of CCK-8, the proliferation outweighs the apoptosis causing hyperplasia but in the case of intermittent administration the opposite effect is seen.     

Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic acini isolated from rats given an ethanol diet

Effects of chronic alcohol intake on secretion and synthesis of amylase and cationic trypsinogen (CT) were studied with pancreatic acini isolated from male Sprague-Dawley rats fed a Lieber-DeCarli ethanol or control diet for 30 days. Pancreatic acini were incubated in a media containing increasing concentrations of cholecystokinin octapeptide (CCK-8: 0-1000pM) followed by addition of [3H]leucine. Amylase and CT secreted in the media and those labeled in acini were quantitated. Basal and CCK-stimulated secretion of CT was not different in alcoholic and control groups. On the other hand, a dose-response curve of CCK-stimulated amylase secretion from alcoholic acini was markedly reduced with both basal and maximal secretion decreased to only 40% of controls. Basal incorporation of [3H]leucine into amylase was reduced by 70% in alcoholic acini compared to controls while that into CT was not different in the two groups. CCK-8 exhibited a biphasic effect on [3H]leucine incorporation into both enzymes in alcoholic acini: low concentrations of CCK-8 (less than 100pM) increased the incorporation whereas high concentrations (greater than 100pM) decreased it. However, in control acini, CCK-8 induced progressive suppression of the incorporation into these enzymes, the pattern of which was similar to that previously observed in fasted rats (Am. J. Physiol. 241:G116-G112, 1981). This difference in the response pattern resulted in significantly higher rates of CCK-8 induced incorporation into CT in alcoholic acini. These results suggest that the differences observed may possibly be attributable not only to alcohol intake but also to the differences in carbohydrate intake and in temporal patterns of diet consumption.     

Biochemical changes in the exocrine pancreas of rats fed caffeine

Coffee consumption has been associated with pancreatic disorders, but the mechanisms involved remain to be elucidated. This investigation examines the effects of caffeine consumption on the structure and function of the exocrine pancreas. Groups of rats, fed ad libitum commercial laboratory diet, were given drinking water which contained either caffeine (0.09 mg/ml) or nothing at all. The rats were allowed drink ad libitum and were killed 6 weeks later. Final body and pancreatic weights were not significantly different between the groups at the end of the experimental period. Although no ultrastructural effects of caffeine on the pancreas were observed, amylase and trypsinogen activity was 35% higher in pancreatic homogenates from caffeine-fed rats compared with controls. In addition, levels of immunoreactive cationic trypsin(ogen) were 41% higher than control levels in pancreases from the caffeine-fed rats. Also, the circulating levels of amylase and immunoreactive cationic trypsin(ogen) in serum were lower in the caffeine group compared with controls. When dispersed pancreatic acini isolated from the caffeine-fed rats were incubated in vitro with increasing concentrations of CCK-8 or nicotine, the rate of release of amylase, trypsinogen, and chymotrypsinogen was lower than in the control rats. This effect did not appear to be due to inhibition of protein synthesis, as determined by [3H]leucine incorporation into acinar protein. These data suggest that prolonged intake of caffeine at common dietary levels inhibits pancreatic enzyme secretion.     

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

Deactivation of ROCK-II by Y-27632 enhances basolateral pancreatic enzyme secretion and acute pancreatitis induced by CCK analogues

In isolated rat pancreatic acini, protein expression of RhoA and Rho-associated kinase, ROCK-II, and the formation of immunocomplex of RhoA with ROCK-II were enhanced by CCK-8, carbachol, and the phorbol ester TPA. The ROCK-specific inhibitor, Y-27632, did not alter basal amylase secretion, whereas it potentiated CCK-stimulated pancreatic enzyme secretion in vitro. During caerulein-induced pancreatitis occurring in mice in vivo, Y-27632 enhanced serum amylase levels and the formation of interstitial edema and vacuolization at 12-18h after the first injection of caerulein. Y-27632 in turn inhibited the recovery of protein expression of ROCK-II at 18h after the first caerulein injection. These results suggest that RhoA and ROCK-II assemble normal CCK-stimulated pancreatic enzyme secretion and prevent caerulein-induced acute pancreatitis.     

Phosphorylation of low molecular mass cytosolic proteins by protein kinase C and protein kinase A in the rabbit exocrine pancreas

Subcellular fractionation of rabbit pancreatic acini was performed to study the distribution of endogenous substrates for protein kinase C. Substrates for protein kinase C were found to be predominantly low molecular mass proteins of cytosolic origin. At least three of these soluble substrates, with molecular masses of 17-19 kDa, were relatively heavily phosphorylated by endogenous as well as purified pancreatic protein kinase C. In the same molecular mass range, 16-18 kDa, soluble proteins were also phosphorylated by protein kinase A. Moreover, addition of cyclic AMP under conditions that activated protein kinase C gave a more than additive labelling of these low molecular mass proteins. The latter observation may be of interest in view of the potentiating effect cyclic-AMP-activated protein kinase A has on amylase secretion stimulated by secretagogues which increase free cytosolic Ca2+ and activate protein kinase C.     

血糖浓度对糖尿病大鼠胰腺外分泌功能的影响

用链佐霉素诱导大鼠产生尿病,其胰腺组织淀粉的含量降低,胆囊收缩素（ＣＣＫ－８）刺激所引起的胰淀粉酶分泌也明显降低。用一种可以降低糖尿病大鼠血糖,但不影响其血清胰岛素水平的药物－－钒酸钠灌胃,可翻转上述变化。体外实验分析表明,高糖可抑制胰腺泡蛋白质的合成,还可以引起胰腺泡膜丙二醛含量增加,从而提示,糖尿病大鼠胰外分泌功能障碍与血液葡萄浓度过高有密切关系。     

Intracellular Ca2+ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini.

The association of 125I-labelled epidermal growth factor (125I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca2+ concentrations. These agents included cholecystokinin-octapeptide (CCK8) and the Ca2+ ionophore A23187. Inhibition by CCK8 was blocked by lowering the incubation temperature from 37 degrees C to 15 degrees C. Moreover, in contrast with studies of intact acini, the binding of 125I-EGF to isolated acinar membrane particles was not affected either by CCK8, or by varying the level of Ca2+ in the incubation medium. These results indicated, therefore, that the inhibition of 125I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37 degrees C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the 125I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca2+ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca2+-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca2+. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca2+ ionophore A23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca2+ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.