thapsigargin and Arteriosclerosis

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.

Topics: Androstadienes; Arteriosclerosis; Blotting, Western; Calcium; Calcium Channels; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Indoles; Macrophages; Monocytes; Myosin-Light-Chain Kinase; Oligonucleotides, Antisense; Phosphorylation; Thapsigargin; Wortmannin

Expression of 15-lipoxygenase (15-LO) is induced over 100-fold in early fatty streak lesions. 15-LO activity leads to the production of specific lipid hydroperoxides, which can have major effects on the expression of proinflammatory genes involved in atherogenesis. We have used retrovirus-mediated gene transfer to achieve stable high expression of 15-LO in human endothelial ECV304 cells. These cells were used to study the effects of 15-LO on the expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), activation of nuclear factor kappa B (NF-kappaB), and T-cell adhesion on endothelial cells. NF-kappaB activation was greatly potentiated by increased 15-LO activity in the stably transduced cells, and both VCAM-1 and ICAM-1 were significantly induced in these cells in response to tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) stimulation, as studied by flow cytometry. The induction of ICAM-1 was sensitive to antioxidants in a dose-dependent manner. The adherence of Jurkat T cells on the 15-LO-expressing endothelial cells was markedly induced after PMA stimulation. These results indicate that 15-LO activity may be involved in the early pathogenesis of atherosclerosis by inducing VCAM-1 and ICAM-1 expression and by increasing T-cell adhesion on the endothelium.

Topics: Antioxidants; Arachidonate 15-Lipoxygenase; Arteriosclerosis; Cell Adhesion; Cell Line; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Genetic Vectors; Humans; Hydrogen Peroxide; Immunoblotting; Intercellular Adhesion Molecule-1; Jurkat Cells; NF-kappa B; Oxidants; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin; Thioctic Acid; Transfection; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1