A possible role for Ca2+/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus-secretion coupling

Ca2+/calmodulin-dependent protein kinases (CaMKs) are important intracellul ar mediators in the mediation of stimulus-secretion coupling and excitation -contraction coupling in a wide variety of cell types. We attempted to iden tify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a Ca MKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaM KII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a r adioenzyme assay (REA) using autocamtide II, peptide gamma and myosin P-lig ht chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide gamma is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carba chol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the c ell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentratio n-dependent increase in CaMKs activities. When autocamtide II was used, max imal increases were 1.5-1.8-fold over basal (20.2 +/- 2.0 pmol/min/mg prote in), with peaks occurring at 20 min after cell stimulation. In separate stu dies that used peptide gamma, CCK-U, CCh and CCK-OPE dose-dependently incre ased 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 s timulation. Peak increases after cell stimulation induced by peptide gamma were 1.8-2.8-fold higher than those induced by autocamtide II. CCK-8, CCh a nd CCK-OPE also significantly increased phosphotransferase activities of my osin light chain kinase (MLCK) substrate (basal: 4.4 +/- 0.7 pmol/min/mg pr otein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide gamma. Characteristics of the phosphotra nsferase activity were also different between autocamtide II and peptide ga mma. When autocamtide II was used, elimination of medium Ca2+ in either cel l lysates or intact cells resulted in a significant decrease in the activit y, whereas it had no or little effect when peptide gamma was used, This sug gests that Ca2+ influx from the extracellular space is not fully required f or CaMKIV activity and Ca2+ is not a prerequisite for phosphotransferase ac tivity once CaMKIV is activated by either intracellular Ca2+ release or int racellular Ca2+ oscillations. The specific CaMKII inhibitor KN-62 (50 mu M) had no effect on the CaMKIV activity and pancreatic enzyme secretion elici ted by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 mu M), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In con trast, wide spectrum CaMK inhibitors, K-252a (1 mu M) and KT5926 (3 mu M), significantly inhibited CaMKIV activities and enzyme secretion evoked by se cretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus-secretion coupling of rat pancreatic acinar cells. (C) 200 0 Elsevier Science B.V, All rights reserved.