Rho-associated Kinase Directly Induces Smooth Muscle Contraction through Myosin Light Chain Phosphorylation

Abstract

Small GTPase Rho plays pivotal roles in the Ca 2؉ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca 2؉ -sensitizing effects in extensively Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor (Gong, M. C., Iizuka, K., Nixon, G., Browne, J. P., Hall, A., Eccleston, J. F., Sugai, M., Kobayashi, S., Somlyo, A. V., and Somlyo, A. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1340 -1345). Here we demonstrate the contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca 2؉ . These effects of constitutively active Rho-kinase were wortmannin (a potent myosin light chain kinase inhibitor)-insensitive. Immunoblot analysis revealed that the amount of native Rho-kinase was markedly lower in Triton X-100-permeabilized tissue than in intact tissue. Our results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independently of the Ca 2؉ -calmodulin-dependent myosin light chain kinase pathway.Smooth muscle contraction is primarily regulated by the levels of phosphorylation of myosin light chain (MLC), 1 which has heretofore been considered to be governed by a Ca 2ϩ -calmodulin (CaM)-dependent MLC kinase pathway (1-4). However, as the use of Ca 2ϩ indicator revealed that the force/ Ca 2ϩ ratio is variable, the Ca 2ϩ -CaM-dependent MLC kinase pathway cannot solely account for the mechanisms of agonistor GTP␥S-induced increases in the force/Ca 2ϩ ratio, so-called Ca 2ϩ sensitization (1,(5)(6)(7)(8)(9). Thus, an additional mechanism that can regulate Ca 2ϩ sensitization of smooth muscle has been proposed. Using membrane permeabilization of smooth muscle, the possibility that monomeric Ras family G-proteins, such as Rho, contribute to Ca 2ϩ sensitization of smooth muscle was demonstrated (10 -12). Direct activation of G-proteins by the application of GTP␥S (8, 9), agonists (1,(5)(6)(7)(8), and GTP-activated Rho (10 -12) could exert Ca 2ϩ -sensitizing effects on saponin-or ␤-escin-permeabilized smooth muscle. However, the activated Rho failed to induce Ca 2ϩ sensitization of extensively Triton X-100-permeabilized smooth muscle (11). Considering that extensive Triton X-100-permeabilization allows higher molecular weight compounds to diffuse from the cytosol of smooth muscle of the rabbit portal vein (13), important diffusible factor(s) for the Ca 2ϩ sensitization of smooth muscle might be lost during extensive permeabilization by Triton X-100, an event that would result in no response to activated Rho.We have recently reported that Rho-kinase, which is activated by GTP-bound act...

Endothelial dysfunction and hypertension in aging

“…Several investigators, including us, have demonstrated that there is a possible association of ROCK activity with oxidative stress and that smoking enhances the activation of ROCKs in vascular smooth muscle cells in vivo and in vitro. 98,109,112 These findings suggest a correlation between oxidative stress and ROCK activity in humans. Hernandez-Perera et al 113 showed that Rho is required for the basal expression of preproendothelin-1 in vascular endothelial cells, which gives rise to endothelin-1.…”

“…ROCK activates myosin light-chain kinase by phosphorylation of the myosin-binding subunit in myosin light-chain phosphatase, leading to contraction of vascular smooth muscle cells. 97,98 Indeed, the RhoA/ROCK pathway has been shown to be involved in the formation of atherosclerotic lesions, vasoconstriction and myocardial hypertrophy and to be activated in patients with hypertension and in patients with coronary artery disease. [99][100][101][102][103][104] Recently, we have shown that FMD is an independent predictor of leukocyte ROCK activity in healthy men and men with cardiovascular risk factors but without established cardiovascular or cerebrovascular diseases, suggesting that cumulative cardiovascular risk, including aging and hypertension, may enhance ROCK activity and endothelial dysfunction.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Signaling through its effectors Rho kinase (ROCK) and mDia, Rho stimulates actin stress fiber formation by stimulating the monomeric actin binding protein profilin, and stabilizes stress fibers by inhibiting the activity of the actin depolymerizing protein, cofilin [35][36][37][38][39][40]. Bundling of actin filaments with myosin to form stress fibers, itself stimulated by ROCK and myosin light chain kinase, stimulates cell contraction and stabilization of focal adhesions [41][42][43][44], points of integrin-extracellular matrix contact between the ventral surface of the cell and the underlying substrate. Rho-mediated contractility is important for retraction of the cell rear and translocation of the cell body [26].…”

“…Activation of Rho stimulates contractility and focal adhesion formation by promoting bundling of actin filaments and associated myosin motors into stress fibers [44]. Rho-ROCK signaling influences contraction through effects on both the myosin and actin: ROCK elevates myosin light chain phosphorylation by inhibiting myosin phosphatase and/or by directly phosphorylating myosin light chain (MLC) [41][42][43]. ROCK also phosphorylates and activates LIM kinase [64], which in turn phosphorylates and inactivates the actin severing protein, cofilin [36,65].…”

Molecular and cellular mechanisms of action of angiotensin II (AT1) receptors in vascular smooth muscle

“…Phosphorylation of the myosin regulatory light chain (MLC 20 ) by MLCK allows the actin-myosin interaction to proceed with resultant ATP hydrolysis, crossbridge formation and force generation. 12 The sensitivity of the contractile process to [Ca 2+ ] i can also be influenced by contractile agonists, probably through modulation of myosin phosphatase activity 13 or by mitogen activated kinase (MAP kinase) dependent phosphorylation of thin filament proteins caldesmon and calponin, 14 and there is evidence that AII can enhance ␣ 1 -adrenoceptor mediated contraction in rabbit aorta by activation of protein kinase C (PKC). 15,16 Surprisingly AII has also been reported to induce a reduction in response to potassium in rabbit mesenteric arteries by a mechanism dependent on a transcriptional process.…”