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 DGK epsilon in vascular smooth muscle cells were investigated. mRNA expression for DGK alpha, DGK epsilon, and DGK zeta 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 DGK epsilon in a filamentous pattern, parallel to the long axis of cell, and on actin stress fibers as shown by doublestaining with fluorescent phalloidin. DGK alpha was detected sparsely in the cytoplasm and nucleus, and DGK zeta was observed as a granular pattern in the nucleus. In order to elucidate the functional significance of DGK epsilon, 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, DGK epsilon was detected diffusely in the cytoplasm without a filamentous stress fiber pattern. Protein and mRNA expression of DGKe in RASMCs was significantly increased by stimulation with serotonin. Inhibition of Rhoassociated kinases by Y-27632 or inhibition of actin polymerization by cytochalasin B resulted in a decrease in the intensity of DGKe immunoreactivity on stress fibers. The results suggest that DGKe interacts with actin stress fibers and is involved in their stability in vascular smooth muscle cells.