Localization of diacylglycerol kinase ε on stress fibers in vascular smooth muscle cells

The expression pattern of diacylglycerol kinase (DGK) and the biological significance of DGKε in vascular smooth muscle cells were investigated. mRNA expression for DGKα, DGKε, and DGKζ was detected in isolated rat aortic smooth muscle cells (RASMCs) and A7r5 cells by reverse transcription with polymerase chain reaction analysis. An immunocytochemical study revealed intense DGKε in a filamentous pattern, parallel to the long axis of cell, and on actin stress fibers as shown by double-staining with fluorescent phalloidin. DGKα was detected sparsely in the cytoplasm and nucleus, and DGKζ was observed as a granular pattern in the nucleus. In order to elucidate the functional significance of DGKε, its immunoreactivity was examined in RASMCs incubated with serotonin, a vasoconstrictive agonist. When RASMCs were stimulated with serotonin, the cells lost their polarization and shortened, i.e., contracted. In RASMCs contracted by serotonin, DGKε was detected diffusely in the cytoplasm without a filamentous stress fiber pattern. Protein and mRNA expression of DGKε in RASMCs was significantly increased by stimulation with serotonin. Inhibition of Rho-associated kinases by Y-27632 or inhibition of actin polymerization by cytochalasin B resulted in a decrease in the intensity of DGKε immunoreactivity on stress fibers. The results suggest that DGKε interacts with actin stress fibers and is involved in their stability in vascular smooth muscle cells.