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, 38 (8), 930-5

Protein Kinase C Delta-Mediated Processes in cholecystokinin-8-stimulated Pancreatic Acini

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Protein Kinase C Delta-Mediated Processes in cholecystokinin-8-stimulated Pancreatic Acini

Edwin C Thrower et al. Pancreas.

Abstract

Objectives: To define the role of protein kinase C delta (PKC delta) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model.

Methods: Pancreatic acinar cells were isolated from (1) rat, and pretreated with a PKC delta-specific inhibitor or (2) PKC delta-deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nmol/L) and cell responses were measured.

Results: The PKC delta inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. Cholecystokinin-8 stimulation induced a typical biphasic dose-response curve for amylase secretion in acinar cells isolated from both PKC delta(-/-) and wild type mice, with maximal stimulation at 10-pmol/L CCK. Cholecystokinin-8 (100 nmol/L) induced zymogen and nuclear factor kappaB activation in both PKC delta(-/-) and wild type mice, although it was up to 50% less in PKC delta(-/-).

Conclusions: In contrast to previous studies, this study has used specific and complementary approaches to examine PKC delta-mediated acinar cell responses. We could not confirm that it mediates amylase release but corroborated its role in the early stages of acute pancreatitis.

Figures

Figure 1
Figure 1. Rottlerin but not GF109203X affect CCK-induced amylase release
Isolated acinar cells were pre-incubated with or without broad-spectrum PKC inhibitors GF109203X and rottlerin (10 μM) for 2 hours, followed by 30 min CCK treatment (0.1nM and 100nM). Amylase release is expressed as % total release [medium/(medium + cells)]. N=3; *p<0.05
Figure 2
Figure 2. Isoform-specific PKC δ translocation inhibitor does not affect CCK-induced amylase release
Isolated acinar cells were pre-incubated with or without PKC δ translocation inhibitor (10 μM) for 2 hours, followed by a) 30 min CCK treatment over a concentration range (0.001nM - 100nM); b) 100 nM CCK treatment at set time points (0, 5, 15, 30 min). Amylase release is expressed as % total release [medium/(medium + cells)]. N=3.
Figure 2
Figure 2. Isoform-specific PKC δ translocation inhibitor does not affect CCK-induced amylase release
Isolated acinar cells were pre-incubated with or without PKC δ translocation inhibitor (10 μM) for 2 hours, followed by a) 30 min CCK treatment over a concentration range (0.001nM - 100nM); b) 100 nM CCK treatment at set time points (0, 5, 15, 30 min). Amylase release is expressed as % total release [medium/(medium + cells)]. N=3.
Figure 3
Figure 3. Genetic deletion of PKC δ does not affect expression levels of other PKC isoforms or production of digestive enzymes in mouse acinar cells
Pancreata from PKC δ -/- and PKC δ +/+ were removed and protein extracts processed for Western blot analysis. Blots were probed with antibodies for A) PKC α, δ, ε and ζ isoforms and B) amylase, trypsinogen and GAPDH; representative blots are shown. These results confirm the absence of PKC δ in the knockout animals and that genetic deletion of PKC δ did not affect the expression of other PKC isoforms or the production of digestive enzymes.
Figure 4
Figure 4. CCK- and carbachol-induced amylase release is not reduced in PKCδ -/- mice versus wild type (PKCδ +/+)
Pancreatic acinar cells were isolated from PKC δ -/- and +/+ mice and then stimulated for 30 min with a) CCK (0.001-100 nM); b) carbachol (0.01-100 μM). Amylase release is expressed as % total release [medium/(medium + cells)]. N=5.
Figure 4
Figure 4. CCK- and carbachol-induced amylase release is not reduced in PKCδ -/- mice versus wild type (PKCδ +/+)
Pancreatic acinar cells were isolated from PKC δ -/- and +/+ mice and then stimulated for 30 min with a) CCK (0.001-100 nM); b) carbachol (0.01-100 μM). Amylase release is expressed as % total release [medium/(medium + cells)]. N=5.
Figure 5
Figure 5. CCK-induced trypsinogen activation is reduced in PKCδ -/- mice versus wild type (PKCδ +/+)
Pancreatic acinar cells were isolated from PKC δ -/- and +/+ animals and then stimulated for 30 min with 100 nM CCK. Trypsin activity is normalized to amylase content and expressed as fold vs. control. N=6. A reduction in trypsin activity is seen in the knockout animals approaching significance.
Figure 6
Figure 6. CCK-induced NFκB activation is reduced in PKCδ -/- mice versus wild type (PKCδ +/+)
Pancreatic acinar cells were isolated from PKC δ -/- and +/+ animals and then stimulated for 30 min with 100 nM CCK. A) NFκB binding activity was measured in nuclear extracts by EMSA. B) NFκB band intensities were quantified in the PhosphorImager and normalized on the band intensity in the corresponding unstimulated control acini. Values are means +/-SEM (N= 5).

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