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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:509.)
© 2001 American Heart Association, Inc.

Vascular Biology

Myosin Light Chain Kinase Regulates Capacitative Ca²⁺ Entry in Human Monocytes/Macrophages

Quang-Kim Tran; Hiroshi Watanabe; Hong-Yen Le; Ling Pan; Minoru Seto; Kazuhiko Takeuchi; Kyoichi Ohashi

From the Departments of Internal Medicine III (Q.-K.T., L.P., K.T.) and Clinical Pharmacology and Therapeutics (H.W., H.-Y.L., K.O.), Hamamatsu University School of Medicine, Hamamatsu, Japan, and Life Science Center (M.S.), Asahi Chemical Industries, Co Ltd, Fuji City, Shizuoka, Japan.

Correspondence to Hiroshi Watanabe, MD, PhD, Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan. E-mail hwat{at}hama-med.ac.jp

Abstract—Monocytes/macrophages are present in all stages of atherosclerosis. Although many of their activities depend to various extents on changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), mechanisms regulating [Ca²⁺]_i in these cells remain unclear. We aimed to explore the role of myosin light chain kinase (MLCK) in Ca²⁺ signaling in freshly isolated human monocytes/macrophages. Large capacitative Ca²⁺ 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 µmol/L) prevented CCE and completely did so at 100 µmol/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 µmol/L) and wortmannin (100 µmol/L). ML-9 also dose-dependently (1 to 100 µmol/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 Ca²⁺-dependent activities of monocytes/macrophages.

Key Words: monocytes/macrophages • capacitative Ca²⁺ entry • myosin light chain kinase

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