Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.

Small GTPase Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho failed to exert Ca2+-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 Ca2+. 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 Ca2+-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 2ϩcalmodulin (CaM)-dependent MLC kinase pathway (1)(2)(3)(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 active form of Rho (14 -16), phosphorylates not only MLC, thereby activating myosin ATPase (17), but also myosin phosphatase, thus inactivating it in vitro (18). These findings in a cell-free system, plus the previous reports of G-protein-mediating Ca 2ϩ sensitization as described above, suggest that Rho-kinase may induce contraction and concomitant MLC phosphorylation of the smooth muscle. We examined the effects of the constitutively active form of Rho-kinase on smooth muscle extensively permeabilized by Triton X-100 and attempted to determine if Rho-kinase would be the factor lost during extensive Triton X-100 permeabilization.

EXPERIMENTAL PROCEDURES
Materials and Chemicals-The catalytic subunit of recombinant Rho-kinase (CAT; molecular mass is about 80 kDa) was expressed as a glutathione S-transferase fusion protein and purified using a baculovirus system and a glutathione-Sepharose column (17). The kinase activity of the elute was determined by phosphorylation assay using S6 peptide as a substrate (15) in buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 100 M ATP (0.5-20 Gbq/ mmol). CaM was purified from bovine brain by previously described method (19). Other materials and chemicals were obtained from commercial sources.
Force Measurement of the Triton X-100-permeabilized Smooth Muscle of the Rabbit Portal Vein-Small strips of male rabbit (2-2.5 kg) portal veins were manually dissected (50 -100 m wide and 0.5-1 mm long), connected to an isometric force transducer (UL-2GR, Minebea, Japan), and mounted in a well (200 l) on a bubble plate (6). After recording contractions evoked by 118 mM K ϩ , the strips were incubated in relaxing solution, followed by 0.5% Triton X-100 for 20 min at 25°C. The solutions have been described in detail elsewhere (6). CaM (0.5 M) was added to all reactive solutions for experiments with chemical permeabilization.
Immunoblot Analysis of Rho-kinase and MLC-0.5% Triton X-100-* This work was supported in part by grants-in-aid for Scientific Research and for Cancer Research, by a grant for Research and Education in Yamaguchi University from the Ministry of Education, Science, Sports, and Culture, Japan, and by grants from the Naito Foundation, the Research Project on Cerebral Vasospasm from Mie University School of Medicine, and the Mie Medical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Measurement of the Extent of MLC Phosphorylation-After treatment with 5.1 g/ml (0.06 M) CAT and/or 10 M wortmannin, the fringe-like strips of the rabbit portal veins permeabilized by Triton X-100 were quickly placed in a frozen slurry of acetone containing 10% trichloroacetic acid and 10 mM DTT to terminate the contractile responses. After depletion of trichloroacetic acid, the strips were homogenized in urea sample buffer containing 20 mM Tris base, 22 mM glycine, pH 8.6, 8 M urea, 10 mM DTT, 10% sucrose, and 0.1% bromphenol blue. The extracts were subjected to glycerol-urea polyacrylamide gel electrophoresis following immunoblotting using anti-MLC antibodies as documented (22). Immunostained proteins were visualized colorimetrically with 4-chloro-1-naphthol and subjected to densitometrical quantitation.

RESULTS AND DISCUSSION
Introduction of CAT, which is not only the constitutively active form of Rho-kinase but also the highly homologous domain among rat (14), bovine (15), human (16), and mouse Rho-associated kinases (23), into the cytosol of the extensively Triton X-100-permeabilized rabbit portal vein smooth muscle provoked a contraction both at a constant cytosolic Ca 2ϩ (pCa 6.5; Fig. 1a) and at a nominally zero cytosolic Ca 2ϩ buffered with 10 mM EGTA (pCa Ͻ Ͻ 8.0; Fig. 1c). CAT exerted contraction, whereas the vehicle had no effect on the force (Fig. 1, a  and c). These contractions were completely reversed by wash out of CAT, contrary to those induced by 10 M microcystin-LR (24, 25) (Fig. 1b). In the absence of cytosolic Ca 2ϩ at pCa Ͻ Ͻ 8.0, the CAT-induced contraction was also reversible (data not shown). In neither intact nor ␣-toxin-permeabilized strips of the portal vein did CAT exert the contractile effects (data not shown). These observations are interpreted to mean that constitutively active CAT could be introduced into the cytosol of the smooth muscle only by extensive membrane permeabilization to induce a reversible contraction.
MLC phosphorylation mediated by Ca 2ϩ -CaM-dependent MLC kinase pathway plays a primary role in smooth muscle contraction through myosin-actin-interaction and the consequent activation of myosin ATPase (2)(3)(4). To investigate involvement of Ca 2ϩ -CaM-dependent MLC kinase pathway in the CAT-induced contraction, we examined the effects of wortmannin (WM), a potent MLC kinase inhibitor (26) on force development induced by cumulative application of CAT (Fig. 2). In the presence of cytosolic Ca 2ϩ at pCa 6.5, in which Ca 2ϩ -CaM-dependent MLC kinase should be active, 10 M WM shifted the dose-response curve down and to the right. In the absence of cytosolic Ca 2ϩ at pCa Ͻ Ͻ 8.0, in which MLC kinase would be hardly activated, 10 M WM did not affect CATinduced force development. In the absence of CAT, treatment of 10 M WM completely inhibited the cytosolic Ca 2ϩ -provoked contraction at pCa 6.5, in the Triton X-100-permeabilized fibers. Considering our finding that WM did not affect the activity of CAT up to 100 M in vitro (data not shown), this WMsensitive component of CAT-induced contraction at pCa 6.5 seemed to be due to inhibition of the Ca 2ϩ -provoked contraction through the Ca 2ϩ -CaM-dependent MLC kinase pathway but not related to the CAT-mediated pathway. All these observa-

Rho-kinase and Smooth Muscle Contraction 12258
tions suggest that the CAT-induced contraction of smooth muscle of rabbit portal vein permeabilized by Triton X-100 is modulated independently by the Ca 2ϩ -CaM-dependent MLC kinase pathway.
To clarify whether CAT induces contraction with a concomitant increase in levels of MLC phosphorylation, we examined the effects of CAT on MLC phosphorylation, using immunoblotting with anti-MLC polyclonal antibody (Fig. 3). As shown in lanes 1-3 of Fig. 3a, at pCa Ͻ Ͻ 8.0, monophosphorylation of MLC was detected only in the presence of CAT and was insensitive to 10 M WM (42.77 Ϯ 9.22% of the total amount of immunostained MLC (n ϭ 4) in the absence of WM, 35.95 Ϯ 3.39% (n ϭ 4; p Ͼ 0.05) in the presence of WM, respectively). At pCa 6.5, shown in lanes 4 -6 of Fig. 3a, CAT potentiated the level of monophosphorylation of MLC (60. 33 Ϯ 1.42%, n ϭ 4), which was partially inhibited by 10 M WM (26.05 Ϯ 5.18%, n ϭ 4, p Ͻ 0.01). Based on the statistical analysis and the results in Fig. 2, this WM-sensitive component of CAT-induced MLC phosphorylation at pCa 6.5 also seemed to be due to inhibition of Ca 2ϩ -CaM-dependent MLC kinase activity. These results are consistent with that of counterparts of the effects of CAT on the contractile responses (Fig. 3b). It was concluded that CAT potentiates the contractile response by increasing the extent of monophosphorylation of MLC.
To determine if native Rho-kinase is one of the cofactors diffusible during permeabilization by Triton X-100, we examined the amounts of native Rho-kinase in intact and permeabilized fibers by immunoblot analysis using rabbit polyclonal antibodies against Rho-kinase. To standardize the densitometrical value, the ratio of densitometrical quantification of immunostaining of Rho-kinase to that of MLC was calculated in both intact and permeabilized fibers. As shown in Fig. 4, the amounts of native Rho-kinase in the Triton X-100-permeabilized rabbit portal vein were markedly lower than those in intact tissue (0.06 Ϯ 0.01 (n ϭ 4) for permeabilized sample, 0.95 Ϯ 0.02 (n ϭ 4) for intact sample, respectively), whereas the amounts of the possible cytoskeletal proteins, such as MLC and myosin heavy chain in permeabilized fibers were similar to the counterparts of intact fibers. These results confirm that extensive permeabilization by Triton X-100 allows for the loss of cytosolic proteins, including Rho-kinase, whereas cytoskeletal proteins such as myosin are stable. Based on all of these findings taken together plus evidence that the direct activation of G-proteins did not exert contractile effects on the extensively Triton X-100-permeabilized smooth muscle (11), we consider that Rho-kinase may be a valid candidate for the key molecule in G-protein-mediating smooth muscle contraction and may be the molecule lost during extensive permeabilization by Triton X-100.
We demonstrate here what seems to be the first evidence that Rho-kinase is a direct effector on the contractile apparatus of smooth muscle, independently of the Ca 2ϩ -CaM-dependent MLC kinase pathway. Except for Ca 2ϩ -independent MLC kinase (9, 13), we find no documentation that the exogenous addition of kinases to the cytosol of permeabilized smooth muscle directly exerts contractile responses comparable with findings with CAT. Because the inhibition of myosin phosphatase may possibly be the main mechanism of the G-protein-mediating Ca 2ϩ sensitization of smooth muscle contraction (1,9,24,25,27), the CAT-induced contraction of G-protein-uncoupled smooth muscle permeabilized by Triton X-100 (Fig. 1) may be also mediated by the inhibition of myosin phosphatase. This notion is supported by our previous finding that Rho-kinase inhibited the activity of myosin phosphatase through thiophosphorylation of its myosin-binding subunit in vitro (18). However, at cytosolic zero Ca 2ϩ , microcystin-LR-induced contraction of the permeabilized smooth muscle was reduced by an MLC kinase inhibitor (25), whereas the CAT-induced contraction was insensitive to it (Fig. 2). Such differential sensitivities of the MLC kinase inhibitor to CAT-and myosin phosphatase inhibitior-induced contractions support the idea that myosin phosphatase inhibition alone cannot account for the CAT-induced contraction at the cytosolic zero Ca 2ϩ . Taking this together with our report that Rho-kinase directly provokes the phosphorylation of MLC and activates myosin in vitro (17), we suggest that the mechanism(s) of CAT-induced contraction of Triton X-100-permeabilized rabbit portal vein might be a concomitant monophosphorylation of MLC directly induced by CAT independently of a Ca 2ϩ -CaM-dependent MLC kinase pathway. We propose that Rho-kinase is considered a valid key molecule in G-protein-mediating Ca 2ϩ sensitization of smooth muscle contraction.