EC:2.7.11.13 - FACTA Search

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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated digestive enzyme release following a short-term pancreatic duct obstruction in rats. An in vivo experiment demonstrated that a 6-hour pancreatic duct obstruction reduced digestive enzyme release evoked both by endogenously released cholecystokinin (CCK) due to pancreaticobiliary diversion and by exogenous administration of CCK8. In vitro experiments also showed that pancreatic duct obstruction reduced the maximal CCK8-evoked amylase secretion. Amylase secretion evoked by the calcium ionophore A23187 and by phorbol myristate acetate was also markedly decreased. These data suggest that pancreatic duct obstruction probably interferes with the secretory process downstream of hormone receptor binding, intracellular Ca2+ release and protein kinase C activation.
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PMID:Pancreatic exocrine secretion in short-term pancreatic duct obstruction induced acute pancreatitis in rats: an in vivo and in vitro study. 752 Mar 99

Cholecystokinin stimulates a variety of physiological effects throughout the gastrointestinal tract, including exocrine pancreatic secretion, contraction of gallbladder and smooth muscle throughout the gastrointestinal tract, relaxation of the sphincter of Oddi and inhibition of gastric emptying. To initiate these responses cholecystokinin must first interact with receptors on the plasma membrane of either pancreatic acinar or smooth muscle cells. Following receptor occupation the receptor is coupled to generation of intracellular messengers, such as ions, cyclic nucleotides or derivatives of phospholipid hydrolysis. These intracellular messengers activate effectors, the systems that cause a biological response. This paper uses the exocrine pancreas as a model for cholecystokinin stimulated signal transduction and examines cholecystokinin stimulated mobilization of calcium and the activation of protein kinase C. Calcium and protein kinase C act differently to mediate either the initial or sustained phases of amylase secretion from the pancreas. The activation of protein kinase C and the rise of intracellular free calcium is necessary for the initial phase of secretion, but unimportant for the sustained phase of secretion. Calcium from extracellular sources is necessary for the sustained phase of secretion. The cholecystokinin-stimulated intracellular signaling outlined for the exocrine pancreas also occurs in other tissues for transmitting the signal from the cholecystokinin receptor to the inside of the cell.
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PMID:Cholecystokinin-stimulated intracellular signal transduction pathways. 752 Apr 83

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
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PMID:Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells. 753 8

We examined the interaction among secretagogues that stimulate pepsinogen secretion through different pathways in vivo and in vitro. In in vitro study, a combined administration of secretin and carbachol or cholecystokinin octapeptide (CCK-8) to the culture medium of chief cells potentiated pepsinogen secretion. Moreover, the response induced by carbachol or CCK-8 with forskolin was greater than that with secretin. We examined the interaction among receptor-related second mediators, and found that carbachol- or CCK-8-induced intracellular Ca2+ concentration ([Ca2+]i) increase was not affected by secretin or forskolin. Both these substances, however, significantly reduced secretin-induced cAMP production. On the contrary, CCK-8 significantly increased forskolin-induced cAMP production, while carbachol increased it slightly. Calcium ionophore, A23187, or protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), did not alter secretin- or forskolin-induced cellular cAMP production; and the reductive effect of carbachol or CCK-8 on secretin-induced cAMP production was restored by their competitive antagonists, atropine or lorglumide. EC50 of those antagonists was almost the same value as IC50 on pepsinogen secretion and [Ca2+]i increase. These results indicate that secretin-induced cAMP production is interfered with by receptor related agonists like CCK-8 and carbachol. It may be suggested that there is a kind of "cross-talk," between the adenylate cyclase system, that is, the secretin receptor, and carbachol or CCK-8 receptor. The interactions between secretin and other secretagogues (carbachol, CCK-8, tetragastrin and histamine) were examined using the perfused rat stomach.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction among secretagogues on pepsinogen secretion from rat gastric chief cells. 755 Jan 21

We used a fluorescent derivative of the myristoylated, alanine-rich C kinase substrate (MARCKS) peptide as a probe for protein kinase C (PKC) activation by cholecystokinin-octapeptide (CCK-8) in isolated pancreatic acinar cell pairs. The diffusion of the acrylodan-labelled MARCKS-peptide into the cell interior could be monitored by the increase of fluorescence in the whole-cell patch-clamp configuration. Addition of 10 pM CCK-8 to the bath induced repetitive fluctuations of the fluorescent signal in the time range of 4-5 min. With 1 nM CCK-8 a sustained decrease of the signal was observed. Addition of polymyxin B, a specific inhibitor of PKC activation, to the pipette filling solution suppressed the CCK-8-induced change of fluorescence. The data indicate activation of PKC by CCK-8 in pancreatic acinar cells and could be compared with the previously studied CCK-8-induced gap junction uncoupling.
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PMID:Cholecystokinin-octapeptide affects the fluorescence signal of a single pancreatic acinar cell loaded with the acrylodan-labelled MARCKS peptide, a protein kinase C substrate. 760 34

Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.
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PMID:Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini. 761 6

The contributions of phosphoinositide (PI)- and phosphatidylcholine (PC)-specific phospholipases [PI-specific phospholipase C (PI-PLC), PC-specific phospholipase C (PC-PLC), and phospholipase D (PLD)] to diacylglycerol (DAG) formation and regulation of the enzymes by G proteins, Ca2+, and protein kinase C (PKC) were examined in dispersed intestinal circular and longitudinal muscle cells. DAG formation induced by cholecystokinin was biphasic and paralleled by PKC activity. The initial phase (approximately 1 min) was mediated by PI-PLC in circular muscle cells and by both PI- and PC-PLC in longitudinal muscle cells, whereas the sustained phase was mediated by PC-PLC and PLD in both cell types. PC-PLC activity during the initial phase was identified by rapid formation of the initial products [3H]phosphocholine (5 sec) and [3H]myristate-labeled DAG (approximately 15 sec). PLD activity did not contribute to DAG formation during the initial phase, and PI hydrolysis had no effect on PC-PLC or PLD activity during the initial or sustained phases. PLD activity during the sustained phase was evident by the formation of [3H]phosphatidylethanol, a PLD-specific transphosphatidylation product. Dephosphorylation of phosphatidic acid (PA) by phosphatidate phosphohydrolase (PPH) accounted for about 50% of DAG formation; inhibition of PPH activity by propranolol or suppression of PA formation by ethanol inhibited DAG formation by 59-69% and 57-62%, respectively. Residual DAG in the presence of ethanol was augmented 55-57% by DAG kinase inhibitor, whereas residual PA was inhibited by 60-67%, implying that PA was derived from DAG, and DAG from PLC-mediated PC hydrolysis. In the presence of ethanol, calphostin C inhibited phosphatidylethanol formation but had no effect on PA or DAG levels, implying that only PLD activity was modulated by PKC. Maintenance of resting intracellular Ca2+ concentrations, rather than an agonist-induced increase in the intracellular Ca2+ concentration, was required for optimal PC-PLC and PLD activity. Guanosine-5'-O-(beta-thio)diphosphate abolished DAG and PA formation in reversibly permeabilized muscle cells. We conclude that DAG formation in intestinal muscle is mediated by time-dependent activation of three phospholipases (PI-PLC, PC-PLC, and PLD) and two converting enzymes (DAG kinase and PPH). PC-PLC and PLD are Ca2+ dependent and appear to be G protein coupled; only PLD is PKC sensitive.
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PMID:Agonist-mediated activation of phosphatidylcholine-specific phospholipase C and D in intestinal smooth muscle. 765 63

We examined the effects of FK506 on amylase secretion and intracellular pathway in stimulus secretion coupling in isolated pancreatic acini. Amylase release from isolated acini in response to cholecystokinin octapeptide (CCK-8) was suppressed by exposing acini to FK506. The suppressing effect of FK506 on amylase release from acini was dependent on the concentration of FK506 and the time of exposure to FK506. Amylase releases in response to 8-bromo-cAMP and vasoactive intestinal peptide and phorbol ester (12-O-tetradecanoylphorbol 13-acetate) were not suppressed when acini were exposed to FK506, suggesting that the cAMP-dependent protein kinase pathway and protein kinase C pathway were not affected by FK506. However, amylase release in response to calcium ionophore (4-bromo-A23187) was suppressed by FK506, whereas the increase in cytosolic free calcium concentration caused by 4-bromo-A23187 was not affected by FK506. These results suggest that FK506 suppressed secretagogue-stimulated amylase release in acini by altering Ca(++)-mediated intracellular pathways. Further, the property of CCK-8 binding site, CCK-8 stimulated inositol phosphates formation, rises in cytosolic free calcium concentration, and calcium efflux from acini were not suppressed by exposing acini to FK506. These findings indicate that FK506 suppresses amylase release by affecting postreceptor intracellular pathways that are mediated by Ca++ in stimulus secretion coupling.
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PMID:Biochemical characterization of the effects of FK506 on signal transduction in exocytotic function of rat pancreatic acini. 767 38

Cholecystokinin (CCK) is a gastrointestinal hormone that acts through a G protein-coupled receptor to stimulate pancreatic enzyme secretion. In this work, we demonstrate that CCK stimulation of dispersed pancreatic acini results in increased tyrosine phosphorylation of several cellular proteins. This is mediated via a calcium-dependent pathway, also activated by a phenethyl ester analogue of CCK and calcium ionophores, and by a protein kinase C-dependent cascade, also activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. All demonstrable stimulated tyrosine phosphorylation events were inhibited by genistein, with different subsets of proteins affected by staurosporine and H-7. The importance of tyrosine phosphorylation events in agonist-stimulated amylase secretion was studied using genistein and staurosporine as protein kinase inhibitors. Genistein inhibited the secretory response to CCK, its phenethyl ester analogue, and calcium ionophores, all known to stimulate secretion in a calcium-dependent fashion. In contrast, genistein had no effect on the secretory response to 12-O-tetradecanoylphorbol-13-acetate, suggesting that the protein kinase C-dependent tyrosine phosphorylation events were not involved in the secretory mechanism. Furthermore, CCK-induced secretion was not affected by relatively specific protein kinase C inhibition by H-7, but was decreased by staurosporine, an inhibitor of both protein kinase C and tyrosine kinase activities in these cells. These results provide evidence that acinar cell tyrosine phosphorylation is stimulated by agonists acting via calcium-dependent and protein kinase C-dependent pathways, with only the calcium-dependent tyrosine phosphorylation cascade involved in triggering hormone-induced amylase release.
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PMID:A role for cholecystokinin-stimulated protein tyrosine phosphorylation in regulated secretion by the pancreatic acinar cell. 768 71

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokinin-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 microM staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state. 769 87

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