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

AbstractCa2+/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 Ca2+ 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 Ca2+ influx from the extracellular space is not fully required for CaMKIV activity and Ca2+ is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca2+ release or intracellular Ca2+ 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

Liquid Chromatography Assay for Routine Monitoring of Cellular Ribavirin Levels in Blood

Ribavirin-induced hemolytic anemia is one cause for cessation of combination therapy with alpha interferon 2b and ribavirin for hepatitis C infection. Determining cellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, because the metabolites accumulate in erythrocytes. We simplified an assay method developed previously to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a sixfold volume of ice-cold distilled water was subjected to acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated by phenyl boronic acid column extraction, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations of 5.3 to 1,024 μM (r(2) = 0.9999). Validation coefficients of variation for intra- and interday assays were 2.9 to 5.8% and 4.3 to 8.3%, respectively. We tested this method by monitoring blood ribavirin concentrations in two hepatitis C patients receiving alpha interferon 2b-plus-ribavirin combination therapy