A novel protein kinase D inhibitor attenuates early events of experimental pancreatitis in isolated rat acini

Abstract

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.