Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism.
Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.
Tapia
J A
JA
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Jensen
R T
RT
García-Marín
L J
LJ
D000098:Acetophenones / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000681:Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002766:Cholecystokinin / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D046790:Pancreas, Exocrine / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D043886:Receptor, Cholecystokinin A / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014443:Tyrosine / Q000378:metabolism*
D000098:Acetophenones; D001578:Benzopyrans; D010446:Peptide Fragments; D020033:Protein Isoforms; D043886:Receptor, Cholecystokinin A; C410885:cholecystokinin 8; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D014443:Tyrosine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D002766:Cholecystokinin; C085746:rottlerin; D051417:Focal Adhesion Kinase 1; D051745:Protein Kinase C-delta; D020928:Mitogen-Activated Protein Kinases; D000681:Amylases