A possible role for Ca(2+)/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus-secretion coupling.
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 gamma and myosin P-light 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, 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 gamma, 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 gamma 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 gamma. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide gamma. 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 gamma 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 microM) 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 microM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 microM) and KT5926 (3 microM), 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.
Yoshida
H
H
Department of Internal Medicine, MSRB I, #6510B, Box 0682, University of Michigan, Ann Arbor, MI 48109, USA.
Nozu
F
F
Lankisch
T O
TO
Mitamura
K
K
Owyang
C
C
Tsunoda
Y
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000681:Amylases / Q000378:metabolism; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D026121:Indole Alkaloids; D008297:Male; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012844:Sincalide / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
D001381:Azepines; D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D013449:Sulfonamides; C056218:ML 9; C063302:KN 62; C017967:W 7; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C049985:staurosporine aglycone; D010770:Phosphotransferases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat; D009219:Myosin-Light-Chain Kinase; D000681:Amylases; D012844:Sincalide; D002118:Calcium