Abstract-There is now considerable evidence supporting a mitogenic action of serotonin (5-HT) on vascular smooth muscle cells (SMC) that might participate in pulmonary hypertension (PH). Our previous studies have demonstrated that 5-HT-induced proliferation depends on the generation of reactive oxygen species and activation of extracellular signal-regulated kinase (ERK) 1/ERK2. Activation of Rho kinase (ROCK) in SMC also may be important in PH. We undertook the present study to assess the role of Rho A/ROCK and its possible relation to ERK1/ERK2 in 5-HT-induced pulmonary artery SMC proliferation. We found that this stimulation of SMC proliferation requires Rho A/ROCK as inhibition with Y27632, a ROCK inhibitor, or dominant negative (DN) mutant Rho A blocks 5-HT-induced proliferation, cyclin D1 expression, phosphorylation of Elk, and the DNA binding of transcription factors, Egr-1 and GATA-4. 5-HT activated ROCK, and the activation was blocked by GR 55562 and GR127935, 5-HT 1B/1D receptor antagonists, but not by serotonin transport ( Key Words: smooth muscle cells Ⅲ serotonin Ⅲ Rho kinase Ⅲ ERK1/ERK2 Ⅲ pulmonary hypertension I n addition to its actions as a vasoconstrictor and neurotransmitter, serotonin (5-HT) is now recognized to be a cellular mitogen. [1][2][3] There is evidence that this mitogenic action is initiated by active transport via a cell surface transporter (SERT) of bovine, rat, and human pulmonary vascular smooth muscle cells (SMC). 1,4 -6 For other cells, the mitogenic action might be started through 1 or more of the cell surface receptors for 5-HT. A hierarchy of cell signaling responses occurs subsequent to ligation of the cell surface transporter or receptor. It has been well-established that these signaling responses include sequential activations of the small GTPase coupled protein, Rac-1, NADPH oxidase producing superoxide that is dismutated to H 2 O 2 , and extracellular signal-regulated kinase (ERK) 1/ERK2 MAP kinase. 2,[7][8][9] The small GTPase Rho A and its effector, Rho kinase (ROCK), also participate in cellular stress fiber formation and cell cycle progression. 10 -19 There has been limited study of the relationship of Rho A and ROCK to 5-HT. One study showed that Rho A bound to GTP is elevated in the rabbit aortic vascular ring preparation treated with 5-HT. 20 Another study suggested the activation of Rho A and ROCK in 5-HT-induced contraction of the bovine middle cerebral artery. 21 Serotonin participates in pulmonary hypertension, 5,22,23 and a polymorphism of the 5-HT transporter has been proposed to be involved in pulmonary hypertension in humans. 24 Because agents that block ROCK are currently available 25,26 and may be useful in pulmonary hypertension, 27-29 we have undertaken an investigation of the potential participation of ROCK in pulmonary arterial SMC signaling and proliferation produced by 5-HT. The results of our study show that ROCK is activated by 5-HT and that its activation is essential for SMC proliferation produced by 5-HT. Furthermore, with the use of a chemi...