Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

In the present study we partially purified calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from pancreatic acinar cells of the guinea pig using diethylaminoethylcellulose and Sephadex G-150 chromatography and characterized the dependence of the enzyme on calcium, phospholipids, diacylglycerol (diolein), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The enriched preparation of protein kinase C contained no cyclic nucleotide-dependent or calcium-dependent, calmodulin-dependent protein kinase activity. The values of Km for H1-histone and ATP were 0.74 +/- 0.22 and 13.1 +/- 3.2 microM, respectively. Pancreatic protein kinase C demonstrated an absolute requirement for calcium and phospholipid for its activation, and diolein or TPA increased the affinity of the enzyme for calcium by 10-fold. With phosphatidylserine the calcium concentration that caused a half-maximal activation (Ka) was 74 +/- 17 microM, whereas with phosphatidylserine and diolein or TPA the Ka for calcium was 7.9 +/- 1.6 or 6.8 +/- 1.3 microM, respectively. Adding phosphatidylethanolamine and phosphatidylserine decreased the Ka for calcium to 2.0 +/- 0.9 microM with diolein and to 0.7 +/- 0.4 microM with TPA. Activation of protein kinase C by TPA and diolein was identical with calcium concentrations greater than 1 microM, but at low calcium concentrations (less than 1 microM) in the presence of phospholipids, maximally effective concentrations of diolein caused only 55% of the activation seen with TPA. In addition to TPA, other phorbol esters such as phorbol dibutyrate and phorbol diacetate, but not phorbol itself, activated protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

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The novel protein kinase C isoforms -δ and -ε modulate caerulein-induced zymogen activation in pancreatic acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00020.2008 ◽

2008 ◽

Vol 294 (6) ◽

pp. G1344-G1353 ◽

Cited By ~ 28

Author(s):

Edwin C. Thrower ◽

Sara Osgood ◽

Christine A. Shugrue ◽

Thomas R. Kolodecik ◽

Anamika M. Chaudhuri ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Acinar Cell ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Cell Fractionation ◽

Zymogen Activation ◽

Cellular Models ◽

Kinase C

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12- O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-δ and PKC-ε reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-δ and -ε, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-δ and -ε isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-ε redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-ε overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-δ and -ε isoforms translocate to specific acinar cell compartments and modulate zymogen activation.

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