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.
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
Edwin C
EC
Thrower
Jingzhen
J
Yuan
Ashar
A
Usmani
Yannan
Y
Liu
Courtney
C
Jones
Samantha N
SN
Minervini
Martine
M
Alexandre
Stephen J
SJ
Pandol
Sushovan
S
Guha
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