Antiproliferative effects of trapidil in vascular smooth muscle cells are associated by inhibition of MAPK and P34cdc2 activity

Trapidil (Tra) is a potent coronary vasodilator. Recent studies have shown that Tra possesses antiproliferative activity in glioma cells and vascular smooth muscle cells (VSMCs). These studies were undertaken to determine the effects of Tra on the cell cycle of cultured rat VSMCs, in particular, the effects on two key enzymes responsible for cell-cycle control in cultured rat VSMCs. VSMCs were synchronized by serum deprivation and then stimulated to enter the cell cycle by serum refeeding; the cell-cycle distribution was subsequently measured by flow cytometry, and VSMCs pretreated with 5, 50, and 500 mu M Tra showed a decrease in S-phase cell-cycle distribution, 13.1, 18.7, and 58.6%, respectively. In addition, the mitotic activity (S + G(2)/M) decreased 12.9, 18.7, and 49.6%, respectively after Tra treatment. The protein expression of p34(cdc2) was determined by Western blot analysis, and Tra treatment did not affect its expression even at 500 mu M Mitogen-activated protein kinase (MAPK) activity and p34(cdc2) kinase activity were assayed by phosphorylation of their specific substrates, myelin basic protein (MBP) and histone H1, after immunoprecipitation. Exposure of VSMCs to Tra resulted in a significant decrease in serum-stimulated MAPK and P34(cdc2) activity. The inhibitory rates of 50 and 500 mu M Tra on MAPK activity were 59.2 and 80.9%, respectively. Tra concentrations of 5, 50, and 500 mu M inhibited p34(cdc2) activity by 16.4, 22.6, and 40.8%, respectively. Furthermore, 500 mu M Tra inhibited the basal kinase activities of MAPK and P34(cdc2) in the cells that were not serum stimulated. These findings demonstrate that Tra significantly decreases the mitotic activity of cultured VSMCs, and that this effect is associated with the inhibitory role of Tra on the kinase activities of MAPK and p34(cdc2).