A novel protein kinase D inhibitor attenuates early events of experimental pancreatitis in isolated rat acini.
Novel protein kinase C isoforms (PKC δ and ε) mediate early events in acute pancreatitis. Protein kinase D (PKD/PKD1) is a convergent point of PKC δ and ε in the signaling pathways triggered through CCK or cholinergic receptors and has been shown to activate the transcription factor NF-κB in acute pancreatitis. For the present study we hypothesized that a newly developed PKD/PKD1 inhibitor, CRT0066101, would prevent the initial events leading to pancreatitis. We pretreated isolated rat pancreatic acinar cells with CRT0066101 and a commercially available inhibitor Gö6976 (10 μM). This was followed by stimulation for 60 min with high concentrations of cholecystokinin (CCK, 0.1 μM), carbachol (CCh, 1 mM), or bombesin (10 μM) to induce initial events of pancreatitis. PKD/PKD1 phosphorylation and activity were measured as well as zymogen activation, amylase secretion, cell injury and NF-κB activation. CRT0066101 dose dependently inhibited secretagogue-induced PKD/PKD1 activation and autophosphorylation at Ser-916 with an IC(50) ∼3.75-5 μM but had no effect on PKC-dependent phosphorylation of the PKD/PKD1 activation loop (Ser-744/748). Furthermore, CRT0066101 reduced secretagogue-induced zymogen activation and amylase secretion. Gö6976 reduced zymogen activation but not amylase secretion. Neither inhibitor affected basal zymogen activation or secretion. CRT0066101 did not affect secretagogue-induced cell injury or changes in cell morphology, but it reduced NF-κB activation by 75% of maximal for CCK- and CCh-stimulated acinar cells. In conclusion, CRT0066101 is a potent and specific PKD family inhibitor. Furthermore, PKD/PKD1 is a potential mediator of zymogen activation, amylase secretion, and NF-κB activation induced by a range of secretagogues in pancreatic acinar cells.
Thrower
Edwin C
EC
Department of Internal Medicine, Section of Digestive Diseases, and Veterans Administration Connecticut Healthcare, West Haven, and Yale University School of Medicine, New Haven, Connecticut, USA. edwin.thrower@yale.edu
Yuan
Jingzhen
J
Usmani
Ashar
A
Liu
Yannan
Y
Jones
Courtney
C
Minervini
Samantha N
SN
Alexandre
Martine
M
Pandol
Stephen J
SJ
Guha
Sushovan
S
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D002766:Cholecystokinin / Q000494:pharmacology; D008297:Male; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010195:Pancreatitis / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
C551536:CRT 0066101; D002227:Carbazoles; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C081021:Go 6976; D002217:Carbachol; D002766:Cholecystokinin; C089212:protein kinase D; D011493:Protein Kinase C; D002401:Cathepsin B