Myosin light chain kinase regulates capacitative ca(2+) entry in human monocytes/macrophages.
Monocytes/macrophages are present in all stages of atherosclerosis. Although many of their activities depend to various extents on changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), mechanisms regulating [Ca(2+)](i) in these cells remain unclear. We aimed to explore the role of myosin light chain kinase (MLCK) in Ca(2+) signaling in freshly isolated human monocytes/macrophages. Large capacitative Ca(2+) entry (CCE) was observed under fura 2 fluoroscopy in human monocytes/macrophages treated with thapsigargin and cyclopiazonic acid. ML-9 and wortmannin, 2 structurally different inhibitors of MLCK, dose-dependently (1 to 100 micromol/L) prevented CCE and completely did so at 100 micromol/L, whereas inhibitors of tyrosine kinase and protein kinase C had only partial effects. Western blotting showed that thapsigargin significantly caused myosin light chain phosphorylation, which was almost completely blocked by ML-9 (100 micromol/L) and wortmannin (100 micromol/L). ML-9 also dose-dependently (1 to 100 micromol/L) inhibited this phosphorylation, which was well correlated with its inhibition of CCE. Transfection with MLCK antisense completely prevented CCE in response to thapsigargin and cyclopiazonic acid, whereas MLCK sense had no effect. These data strongly indicate that MLCK regulates CCE in human monocytes/macrophages. The study suggests a possible involvement of MLCK in many Ca(2+)-dependent activities of monocytes/macrophages.
Tran
Q K
QK
Departments of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Watanabe
H
H
Le
H Y
HY
Pan
L
L
Seto
M
M
Takeuchi
K
K
Ohashi
K
K
D000730:Androstadienes / Q000494:pharmacology; D001161:Arteriosclerosis / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007211:Indoles; D016376:Oligonucleotides, Antisense; D019284:Thapsigargin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; C000543:cyclopiazonic acid; D000077191:Wortmannin