Cyclosporine A Inhibits Protein-Kinase-C-Mediated Amylase Release from Isolated Rat Pancreatic Acini

Digestion ◽  
1994 ◽  
Vol 55 (6) ◽  
pp. 380-388 ◽  
Author(s):  
Michael Höcker ◽  
Ingo H. Waschulewski ◽  
Horst F. Kern ◽  
Klaus A. Domagk ◽  
Rainer Schwarzhoff ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclosporine A ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Isolated Rat

scholarly journals Protein kinase A modulates Ca2+- and protein kinase C-dependent amylase release in permeabilized rat pancreatic acini

1992 ◽  
Vol 287 (2) ◽  
pp. 403-406 ◽  
Author(s):  
A J O'Sullivan ◽  
J D Jamieson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Response Curve ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Pkc Activation ◽  
Kinase A

The role of protein kinase A (PKA) in the release of amylase from permeabilized pancreatic acini was investigated. Addition of cyclic AMP (cAMP) to permeabilized acini resulted in a potentiation of Ca(2+)-dependent amylase release, shifting the Ca2+ dose/response curve leftwards. As with protein kinase C (PKC) activation, this is due to an increase in the time of active discharge. The effect of cAMP was shown to be blocked by two inhibitors of PKA, H89 and the PKI-(5-24)-peptide. At low concentration, cAMP synergizes from phorbol 12-myristate 13-acetate (PMA), while at optimal concentrations cAMP and PMA are additive. PKA and PKC appear to work via similar, but not identical mechanisms.

Effects of staurosporine on protein kinase C and amylase secretion from pancreatic acini

1989 ◽  
Vol 257 (4) ◽  
pp. G548-G553 ◽  
Author(s):  
T. B. Verme ◽  
R. T. Velarde ◽  
R. M. Cunningham ◽  
S. R. Hootman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Digestive Enzyme ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Functional Protein ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity

The effects of staurosporine, a recently isolated microbial alkaloid, on amylase secretion and protein kinase C activity of guinea pig pancreatic acini were investigated. Staurosporine at a concentration of 1 microM completely inhibited both acinar protein kinase C activity (IC50 = 5.5 +/- 1.4 nM) and amylase secretion induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (IC50 = 4.1 +/- 0.4 nM). At this concentration, staurosporine reduced amylase secretion elicited by maximally effective concentrations of carbachol and cholecystokinin by approximately 50% but did not appreciably alter the potencies of the two secretagogues. In the presence of staurosporine, amylase secretion induced by carbachol was linear for at least 60 min. Staurosporine had no effect on amylase release elicited by the Ca2+ ionophore A23187. It did, however, inhibit secretion induced by vasoactive intestinal peptide, although with a reduced potency relative to its effects on amylase release stimulated by TPA, carbachol, and cholecystokinin (IC50 = 34 +/- 17 nM). These results indicate that staurosporine is a potent inhibitor of protein kinase C activity in pancreatic acini and that protein kinase C has an important role as an intracellular mediator of digestive enzyme secretion induced by cholecystokinin and carbachol in the acinar cell. In addition, a separate staurosporine-insensitive coupling pathway, most likely involving Ca2+, appears to be equally important and can maintain long-term secretion in the absence of functional protein kinase C activity.

Carbachol acts through protein kinase C to modulate cholecystokinin receptors on pancreatic acini

1991 ◽  
Vol 261 (6) ◽  
pp. G981-G986 ◽  
Author(s):  
D. S. Louie ◽  
O. Y. Chung
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Capacity ◽  
Muscarinic Agonist ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
High Affinity ◽  
Total Binding ◽  
Kinase C

Cholecystokinin (CCK) and cholinergic agonists are both major stimulants of pancreatic enzyme secretion and both utilize a common calcium-phosphoinositide-mediated receptor coupling system. In this study we investigated the modulation of pancreatic acinar CCK receptors by the muscarinic agonist carbachol (CCh) and investigated the intracellular mechanisms involved in the modulation. Acini were isolated from rat pancreas and dispersed in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Ringer solution. Preincubation with 0.1 mM carbachol for 60 min reduced the CCK octapeptide (CCK-8; 100 pM)-stimulated amylase release by 43 +/- 5%. Binding of 125I-Bolton-Hunter-labeled CCK-8 (125I-BH-CCK-8) revealed two classes of CCK receptors, a high affinity with a dissociation constant (Kd) of 20 pM and a low affinity with a Kd of 2.3 nM. Pretreatment with 100 microM CCh decreased total binding by 35 +/- 6%, affecting the binding capacity of the high-affinity site, without change in the maximal binding capacity of the low-affinity site and no change in the Kd of either site. Preincubation of acini with 12-O-tetradecanoylphorbol 12,13-acetate (TPA, 1 microM), an activator of protein kinase C (PKC), decreased subsequent CCK-8-stimulated amylase release, and total binding of 125I-BH-CCK-8 to a similar extent as with pretreatment with CCh. The inhibitory effect of TPA or CCh on CCK-8-stimulated amylase release was reversed by simultaneous preincubation with H-7, an inhibitor of PKC. Pretreatment of acini with the calcium ionophore A23187, vasoactive intestinal peptide, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on 125I-BH-CCK-8 binding. After CCh or TPA preincubation, CCK-8-stimulated production of [3H]inositol phosphates was inhibited by at least 49%.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

1992 ◽  
Vol 285 (2) ◽  
pp. 597-601 ◽  
Author(s):  
A J O'Sullivan ◽  
J D Jamieson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peptide Inhibitor ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Streptolysin O ◽  
Absolute Requirement ◽  
Messenger System

The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca(2+)-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca(2+)-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-31-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca(2+)-dependent amylase discharge.

Caerulein causes translocation of protein kinase C in rat acini without increasing cytosolic free Ca2+

1988 ◽  
Vol 255 (1) ◽  
pp. G33-G39 ◽  
Author(s):  
R. Bruzzone ◽  
R. Regazzi ◽  
C. B. Wollheim
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Microsomal Fraction ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Enhanced Secretion

We investigated the relationships between changes in cytosolic free Ca2+ ([Ca2+]i) and amylase secretion in dispersed rat pancreatic acini. Although 10 pM caerulein did not raise [Ca2+]i, higher concentrations (1 nM) of the peptide elicited a prompt, marked, but transient (2-3 min) elevation of [Ca2+]i. Both concentrations of caerulein caused an almost identical release of amylase over a 30-min period. To investigate the mechanism(s) underlying Ca2+-independent secretion, we measured the effect of the secretagogue on protein kinase C activity and found that both caerulein concentrations caused a significant translocation of protein kinase C from the cytosolic to the microsomal fraction. Because 1 nM caerulein induced a greater enzyme secretion than 10 pM caerulein during the first 2-5 min of stimulation, we explored further the role of [Ca2+]i transients during the first minutes of secretion. Addition of ionomycin in the presence of 10 pM caerulein resulted in a rise in [Ca2+]i and enhanced secretion as a result of caerulein in a near additive fashion during the first 2 min of stimulation. Second, we pretreated acini for 5 min with 1 microM 12-O-tetradecanoylphorbol-13-acetate. This maneuver inhibited both caerulein-induced inositol trisphosphate formation and [Ca2+]i elevation. These findings were paralleled by a similar inhibition of caerulein-stimulated amylase release only during the first 5 min of secretion. These results indicate that 1) caerulein can stimulate amylase secretion independently of a concomitant [Ca2+]i rise, possibly by activation of protein kinase C, and 2) an elevation of [Ca2+]i serves as a trigger to enhance amylase release only during the initial phase of secretion.

Downregulation of protein kinase C in guinea pig pancreatic acini: effects on secretion

1988 ◽  
Vol 254 (2) ◽  
pp. G242-G248 ◽  
Author(s):  
C. K. Sung ◽  
S. R. Hootman ◽  
E. L. Stuenkel ◽  
C. Kuroiwa ◽  
J. A. Williams
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Guinea Pig ◽  
Ionophore A23187 ◽  
Response Curves ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Almost All

Pretreatment of guinea pig pancreatic acini with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and concentration-dependent down-regulation of protein kinase C. In control acini almost all of the protein kinase C activity was present in a cytosolic fraction. Incubation with TPA initially shifted protein kinase C activity to a particular fraction which then disappeared over the following 24-h incubation with TPA. To study the role of protein kinase C in stimulus-secretion coupling, acini were pretreated with TPA and then amylase release was studied in response to various secretagogues. Preincubation of acini with TPA led to a time- and concentration-dependent decrease in TPA-stimulated amylase release that correlated with protein kinase C downregulation. Preincubation of acini with 1 microM TPA for 24 h, resulting in complete loss of protein kinase C activity, abolished the secretory effect of subsequently added TPA. By contrast, the secretory effects of cholecystokinin octapeptide (CCK-8) and carbamylcholine chloride (CCh) were only inhibited by 44 and 34%, respectively, and amylase release stimulated by the Ca2+ ionophore A23187 and an adenosine 3',5'-cyclic monophosphate-mediated agonist, vasoactive intestinal peptide, was unaffected. Dose-response curves for CCK-8- or CCh-stimulated amylase release in TPA-pretreated acini revealed attenuation of both maximal efficacy and sensitivity. However, the CCh-stimulated intracellular Ca2+ increase as determined by use of the fluorescent probe fura-2 was not affected by the long-term TPA pretreatment of acini. This study strongly suggests that both protein kinase C and intracellular Ca2+ play a significant role in CCK-8- and CCh-stimulated amylase release.

Amylase release from streptolysin O-permeabilized pancreatic acini

1990 ◽  
Vol 259 (2) ◽  
pp. G157-G164 ◽  
Author(s):  
M. Kitagawa ◽  
J. A. Williams ◽  
R. C. De Lisle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein S ◽  
Bacterial Toxin ◽  
Amylase Secretion ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Streptolysin O ◽  
Gamma S

Intracellular mediators of exocytosis were investigated using isolated mouse pancreatic acini permeabilized with the bacterial toxin streptolysin O (SLO). Permeabilization was demonstrated by fluorescent staining with ethidium bromide and fluorescein diacetate and release of cytoplasmic lactate dehydrogenase. When SLO-permeabilized acini were incubated at 37 degrees C in Ca2(+)-EGTA buffers containing MgATP, amylase secretion was Ca2+ dependent with an EC50 of 0.40 microM Ca2+ and a maximally effective Ca2+ concentration of 1 microM. Maximal amylase secretion was 330% of that in Ca2(+)-free buffer (basal). The nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 30 microM) increased the maximal secretion to 451% of basal in the presence of 1 microM Ca2+ and decreased the EC50 to 0.14 microM Ca2+. Removal of ATP plus addition of antimycin A and 2-deoxy-D-glucose inhibited Ca2(+)-dependent, GTP gamma S-enhanced amylase secretion by 56%. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) also enhanced maximal secretion to 450% of basal and decreased the EC50 to 0.18 microM Ca2+. Enhancement of amylase secretion by submaximal concentrations of GTP gamma S or TPA was inhibited by the protein kinase C inhibitor staurosporine. These results suggest that Ca2+ stimulation of amylase secretion is potentiated by activation of protein kinase C. However, the enhancement of secretion by GTP gamma S and TPA was additive at their maximally effective concentrations, suggesting that another G protein(s) maybe involved in the terminal steps of exocytosis.

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