Abstract. Vascular smooth muscle contraction is mediated by activation of extracellular signalregulated kinase (ERK) 1 / 2, an isoform of mitogen-activated protein kinase (MAPK). However, the role of stress-activated protein kinase /c-Jun N-terminal kinase (JNK) in vascular smooth muscle contraction has not been defined. We investigated the role of JNK in the contractile response to norepinephrine (NE) in rat aortic smooth muscle. NE evoked contraction in a dosedependent manner, and this effect was inhibited by the JNK inhibitor SP600125. NE increased the phosphorylation of JNK, which was greater in aortic smooth muscle from hypertensive rats than from normotensive rats. NE-induced JNK phosphorylation was significantly inhibited by SP600125 and the conventional-type PKC (cPKC) inhibitor Gö6976, but not by the Rho kinase inhibitor Y27632 or the phosphatidylinositol 3-kinase inhibitor LY294002. Thymeleatoxin, a selective activator of cPKC, increased JNK phosphorylation, which was inhibited by Gö6976. SP600125 attenuated the phosphorylation of caldesmon, an actin-binding protein whose phosphorylation is increased by NE. These results show that JNK contributes to NE-mediated contraction through phosphorylation of caldesmon in rat aortic smooth muscle, and that this effect is regulated by the PKC pathway, especially cPKC.

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ABSTRACT. We determined the contribution of the Rho family of low molecular GTP-binding proteins to phorbol ester-induced contraction in swine pulmonary artery smooth muscle. In Ca 2+ -free medium containing 1 mM EGTA, 12-deoxyphorbol 13-isobutyrate (DPB, 1 µM), a protein kinase C (PKC) activator, elicited sustained contractions, which were not inhibited by treatment with verapamil, a voltagedependent Ca 2+ channel antagonist, and Y27632, a Rho-associated kinase inhibitor. Immunoblot analysis showed three PKC isoforms (α, ε, and ζ) and two Rho GTPases (RhoA and Cdc42) in both cytosolic and the membrane fractions from quiescent strips. DPB (1 µM) significantly induced PKCα and ε to translocate from the cytosolic to the membrane fraction in Ca 2+ -free medium. DPB also elicited the translocation of Cdc42, but not RhoA to the membrane fraction. Similarly, in the experiment for measurement of Rho GTPase activity by pull-down assay, DPB (1 µM) significantly increased the activity of Cdc42 in Ca 2+ -free medium. Norepinephrine (NE, 10 µM) stimulated the redistribution of RhoA from the cytosolic to the membrane fraction in swine pulmonary artery smooth muscle. In contrast, NE did not alter the subcellular distributions of Cdc42 and the PKC isoforms. These results indicate that phorbol ester evokes PKC-mediated Ca 2+ -independent contraction via a Rho GTPase pathway, especially Cdc42, in smooth muscle from swine pulmonary arteries. KEY WORDS: Ca 2+ -independent contraction, Cdc42, protein kinase C, pulmonary artery smooth muscle.