A possible role for Ca 2+/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus–secretion coupling

Abstract

Ca 2+/calmodulin-dependent protein kinases (CaMKs) are important intracellular mediators in the mediation of stimulus–secretion coupling and excitation–contraction coupling in a wide variety of cell types. We attempted to identify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a CaMKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaMKII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a radioenzyme assay (REA) using autocamtide II, peptide γ and myosin P-light chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide γ is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carbachol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the cell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentration-dependent increase in CaMKs activities. When autocamtide II was used, maximal increases were 1.5–1.8-fold over basal (20.2±2.0 pmol/min/mg protein), with peaks occurring at 20 min after cell stimulation. In separate studies that used peptide γ, CCK-8, CCh and CCK-OPE dose-dependently increased CaMKIV activities. Maximal increases were 1.5–2.4-fold over basal (30.7±3.2 pmol/min/mg protein) with peaks occurring at 20 min after cell stimulation. Peak increases after cell stimulation induced by peptide γ were 1.8–2.8-fold higher than those induced by autocamtide II. CCK-8, CCh and CCK-OPE also significantly increased phosphotransferase activities of myosin light chain kinase (MLCK) substrate (basal: 4.4±0.7 pmol/min/mg protein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide γ. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide γ. When autocamtide II was used, elimination of medium Ca 2+ in either cell lysates or intact cells resulted in a significant decrease in the activity, whereas it had no or little effect when peptide γ was used. This suggests that Ca 2+ influx from the extracellular space is not fully required for CaMKIV activity and Ca 2+ is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca 2+ release or intracellular Ca 2+ oscillations. The specific CaMKII inhibitor KN-62 (50 μM) had no effect on the CaMKIV activity and pancreatic enzyme secretion elicited by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 μM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 μM) and KT5926 (3 μM), significantly inhibited CaMKIV activities and enzyme secretion evoked by secretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus–secretion coupling of rat pancreatic acinar cells.