We have previously reported that 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), a 12-lipoxygenase product of arachidonic acid in platelets, is a potent chemoattractant for rat aortic smooth muscle cells. In the present study, the mechanism involved in 12-HETE-associated smooth muscle cell migration was investigated in relation to calcium mobilization in the cells. Migration of smooth muscle cells was measured by a filter membrane technique in modified Boyden chambers. Smooth muscle cell migration induced by 12-HETE increased with the increase of extracellular Ca2+ concentration and became maximal at the physiological Ca2+ concentration of 1.25 mM. The calcium ionophore A23187, at concentrations of 0.2 and 2.0 μM, significantly stimulated cell migration. Nicardipine, a potent calcium-entry blocker, significantly inhibited 12-HETE-associated smooth muscle cell migration at concentrations from 10-9 to 10-5 M. Concentrations of trifluoperazine from 10-9 to 10-5 M and W-7 at 10-5 M, which are specific inhibitors of calmodulin, also significantly inhibited cell migration induced by 12-HETE. Cytochalasin B at 1.0 and 10 μM, and colchicine at 0.1 and 1.0 μM concentrations drastically inhibited cell migration, indicating that actin-containing microfilaments and microtubules are involved in smooth muscle cell migration. These findings indicated that the stimulation of smooth muscle cell migration by 12-HETE is a highly calcium-dependent process and suggest that 12-HETE might act at the initial stage of smooth muscle cell migration through enhancing calcium influx through plasma membrane and thus stimulating cell migration.
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