Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

Hiromitsu Nagumo1, Yasuharu Sasaki2, Yoshitaka Ono3, Hiroyuki Okamoto1, Minoru Seto2, and Yoh Takuwa4

1 Departments of Molecular and Cellular Physiology and Cardiovascular Biology, University of Tokyo School of Medicine, Tokyo 113-0033; 2 Life Science Center, Asahi Chemical Industry, Fuji 416-0934; 3 Department of Biology, Faculty of Science, Kobe University, Kobe 657-0017; and 4 Department of Physiology, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated inhibition of MLC20 dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgamma S-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20 phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

myosin light chain dephosphorylation; small G protein; calcium ion; sensitization; vascular smooth muscle; contraction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RHO GTPASE, a member of the Rho subgroup of the Ras superfamily, is involved in such diverse biological processes as smooth muscle contraction, cell motility, reorganization of the actin cytoskeleton, cell adhesion, and cell growth (12, 29). Until recently, little was known about the molecular mechanisms by which Rho mediates these activities. A number of Rho target molecules have now been identified, providing much insight into the molecular mechanisms of the Rho actions (2, 20, 25, 34). These include the serine/threonine kinase Rho kinase/ROKalpha /ROCKII (19, 22) and its close relative ROKbeta /ROCKI (13, 19), protein kinase N (PKN), which is another class of serine/threonine protein kinase (18), p140mDia, rhothekin, rhophilin, citron, and citron kinase (2, 20, 25, 34). Among these, the Rho kinase family is of particular interest.

Recent evidence reveals that receptor activation by excitatory agonists in smooth muscle is coupled to activation of a Rho-dependent signaling pathway that leads to inhibition of myosin phosphatase and resultant enhancement of the 20-kDa myosin light chain (MLC20) phosphorylation and of contraction (7-11, 16, 21, 26, 30). It is also shown that, in nonsmooth muscle cells, the receptor agonist stimulation results in a decrease of phosphatase activity in a myosin-rich fraction in a Rho-dependent manner (6). Rho kinase is implicated in Rho inhibition of smooth muscle myosin phosphatase; Rho kinase is shown to be capable of phosphorylating purified smooth muscle myosin phosphatase and consequently inhibiting its activity in vitro (13), and a Rho kinase inhibitor Y-27632 has been demonstrated to inhibit a receptor agonist-induced smooth muscle contraction (7, 33). Smooth muscle myosin phosphatase consists of the 38-kDa catalytic subunit (the protein phosphatase type 1delta isoform), the 130-kDa myosin-binding regulatory subunit (MBS), and the 21-kDa regulatory subunit (M21; see Refs. 1 and 28). MBS, which serves as the targeting subunit of myosin phosphatase to myosin and enhances its activity toward myosin, is a subunit phosphorylated by Rho kinase (15). It has been demonstrated in nonmuscle cells that expression of activated Rho mutant and stimulation with receptor agonists induces an increase in the extent of MBS phosphorylation (15, 24). However, direct evidence that Rho kinase indeed acts downstream of Rho to mediate MBS phosphorylation and myosin phosphatase inhibition in vivo in smooth muscle cells (SMCs) has not yet been found.

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077) has been previously shown to act as a vasodilator in vivo when administered in animals (4) and is currently used for the treatment of cerebral vasospasm. This compound also inhibits agonist-induced contraction of isolated vascular smooth muscle. Agonist stimulation of smooth muscle induces a rise in the intracellular free Ca2+ concentration and the activation of the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK), initiating phosphorylation of MLC20 and contraction (14). HA-1077 inhibits agonist-induced phosphorylation of MLC20 in vascular smooth muscle (27); however, HA-1077 is only a weak inhibitor for isolated MLCK (4), suggesting that HA-1077 has a target other than MLCK in inhibiting MLC20 phosphorylation. We reasoned that HA-1077 might act as an in vivo inhibitor for Rho kinase.

In the present study, we demonstrate that HA-1077 is a potent in vitro inhibitor for purified Rho kinase. We examined in vascular SMCs whether HA-1077 could reverse Rho-mediated myosin phosphatase inhibition and the resultant enhancement of MLC20 phosphorylation. We next examined whether this was accompanied by inhibition of Rho-mediated MBS phosphorylation. The present results reveal that HA-1077 effectively reverses Rho-mediated MBS phosphorylation and myosin phosphatase suppression with a reduction in MLC20 phosphorylation, providing evidence that Rho kinase is a downstream effector of Rho to regulate myosin phosphatase in vivo in SMCs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell culture, permeabilization, and MLC20 phosphorylation. Pig aortic SMCs were obtained as previously described (26) and were used between the 5th and the 15th passages. Before each experiment, the cells were deprived of serum for 24 h. In the experiments of Fig. 4, SMCs freshly isolated by emzymatic digestion of aortic media were seeded onto a culture dish, attached by incubation with 5% serum-containing medium for 5 h, and, after 12 h of serum deprivation, employed for the experiments. Phosphorylation and dephosphorylation of MLC20 in beta -escin-permeabilized SMCs were determined as described in detail previously (26). A percent value of the sum of monophosphorylated and diphosphorylated forms of total MLC20 was calculated.

Rho kinase assay. Rho kinase was purified from bovine brain as described previously (22). To determine the effect of HA-1077 on Rho kinase activity in vitro, the indicated concentrations of HA-1077 were included in 200 µl of the assay mixture containing 50 mM Tris · HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol (DTT), various concentrations of ATP, purified Rho kinase, and 0.1 mg/ml histone HI. Assays were started by the addition of [gamma -32P]ATP. 32P incorporation into histone HI was linear over the initial 5 min. The incubation was performed for 5 min at 30°C and was quenched by the addition of 1 ml of ice-cold 20% TCA followed by the addition of 500 µg of BSA as a carrier protein. After the sample was centrifuged at 3,000 rpm for 15 min, the pellet was resuspended in ice-cold 10% TCA, and the centrifugation-resuspension cycle was repeated three times. The final pellet was dissolved in 1 N NaOH, and the radioactivity was measured by a liquid scintillation counter. The Michaelis-Menten equation was used to calculate the Michaelis constant (Km) and maximal velocity (Vmax) of Rho kinase. Data were further analyzed with a secondary plot (inhibitor concentration vs. Km/Vmax) to calculate the inhibitory constant (Ki) values.

Myosin phosphatase assay. Polyclonal rabbit anti-MBS antibody, anti-PP1delta isoform antibody, and anti-M21 regulatory subunit of myosin phosphatase antibody were raised against the amino-terminal peptide (MKMADAKQKRNE) of chicken MBS and the carboxy-terminal peptide (SGRPVTOORTANPPKKR) of human PP1delta , and recombinant chicken M21 was fused to GST as described (31). For the immunoprecipitation of myosin phosphatase, SMCs were lysed with a lysis buffer containing 60 mM beta -glycerophosphate, 0.5% Nonidet P-40, 0.2% SDS, 100 mM NaF, 1 mM Na3VO4, 2 mM EGTA, 80 µg/ml each of aprotinin and leupeptin, 0.6 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 50 mM Tris · HCl (pH 8.0) and were passed through a 26-G needle five times. After centrifugation at 10,000 g for 5 min, the supernatant was recovered and incubated with anti-MBS antibody at 4°C for 3 h. Immunoprecipitates recovered on protein A-Sepharose (Amersham-Pharmacia Biotechnology) were washed, and then the associated phosphatase activity toward 32P-labeled chicken gizzard MLC20 (1) was measured in vitro in the reaction mixture (100 µl) containing 50 mM Tris (pH 7.5), 4 mM EDTA, 2 mM EGTA, 2 mM DTT, and 10 µM of 32P-labeled chicken gizzard MLC20 at 30°C for 20 min. The reaction was quenched by the addition of 100 µl of ice-cold 20% TCA and 7 µl of 3% BSA. The tubes were left on ice for 15 min and then clarified by centrifugation. The amount of 32P radioactivity released was determined by counting the radioactivity in the supernatant. In preliminary experiments, we found that the amount of 32P radioactivity released showed a linear increase for the first 20 min of the reaction. The amounts of 32P radioactivity released were corrected for the amounts of immunoprecipitated MBS and were expressed as a percentage of the control value.

For Western blotting, immunoprecipitates or cells were solubilized in Laemmli's SDS-sample buffer and resolved by SDS-PAGE (31). Proteins in the gel were electrotransferred to an Immobilon-P membrane (Millipore). After incubation with 3% BSA in Tris-buffered saline [137 mM NaCl and 20 mM Tris · HCl (pH 7.6)] for blocking nonspecific binding of the antibody, the membrane was probed with the respective antibodies, followed by treatment with alkaline phosphatase-conjugated secondary antibody (Zymed).

Phosphorylation of MBS in permeabilized SMCs. Permeabilized SMCs were incubated in the phosphorylation buffer containing 100 µM of [gamma -32P]ATP (50 µCi/ml) for 10 min and were lysed in the lysis buffer. MBS protein in cell lysates was immunoprecipitated as described above and was separated on an 8% SDS-PAGE. The gel was dried and subjected to autoradiography. The radioactivity of the band corresponding to MBS was determined by a Fuji BAS-2000 Bio-Image Analyzer (Fuji, Tokyo, Japan) and was corrected for amounts of MBS.

Plasmids. The cDNA of chicken M21 subunit of myosin phosphatase was cloned by reverse transcription and PCR using total RNA prepared from chicken gizzard. M21 cDNA was ligated into pGEX-2T vector (Amersham-Pharmacia Biotechnology) at the BamH I site, and recombinant GST-M21 fusion protein was produced in the DH5alpha strain of Escherichia coli as described. The cDNA of the pleckstrin homology (PH)/cystein-rich region (amino acids 1124-1388) of the Rho kinase was cloned by reverse transcription and PCR using bovine aortic endothelial cell poly(A)+ RNA. The cDNA for the PH/cystein-rich domain of Rho kinase was subcloned into pQE30 vector (Qiagen) at the BamH I and Hind III sites, and a recombinant hexahistidine-tagged PH/cystein-rich domain was produced in the M15 strain of E. coli. The nucleotide sequences of the cDNAs obtained by the PCR method were confirmed by sequencing with an ALFred DNA sequencer (Amersham-Pharmacia Biotechnology).

Materials. HA-1077 was synthesized by Asahi Chemical Industries. C3 toxin was purchased from Wako (Osaka, Japan). Mouse monoclonal anti-Rho A antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal specific anti-Rho kinase antibody and anti-M21 antibody were raised against the amino-terminal peptide (MSRPPPTGKMPGAP) of bovine Rho kinase and the recombinant GST-M21 fusion protein, as described in Ref. 31. Other chemicals were of reagent grade purity.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We first examined whether or not the protein kinase inhibitor HA-1077 inhibits the activity of purified Rho kinase and found that this was the case. The kinetic analysis (Fig. 1) reveals that HA-1077 acts as a competitive inhibitor versus ATP. The Km value of Rho kinase for ATP is calculated to be 1.2 mM. The Ki values of HA-1077 for Rho kinase and several other serine/threonine protein kinases are compared in Table 1. HA-1077 displays the highest affinity for Rho kinase among the protein kinases examined; its affinity for Rho kinase is 2.5 times higher than for another class of Rho-associated protein kinase (PKN), 5 times higher than for cAMP-dependent protein kinase and cGMP-dependent protein kinase, and 10 times higher than for protein kinase C purified from rat brain. Notably, the affinity of HA-1077 for MLCK is ~100 times lower than that for Rho kinase.


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Fig. 1.   Kinetic analysis of Rho kinase inhibition by 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077). Enzyme activity of Rho kinase purified from bovine brain was measured in the absence and presence of the indicated concentrations of HA-1077 and various amounts of ATP. Competitive inhibition was observed with respect to ATP.


                              
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Table 1.   Ki values of HA1077 for various serine/threonine protein kinases

We then examined how HA-1077 affected the Rho-mediated regulation of MLC20 phosphorylation in vascular SMCs (Fig. 2). In beta -escin-permeabilized SMCs, increasing the ambient free Ca2+ concentration caused a dose-dependent increase in the extent of MLC20 phosphorylation. As we previously reported (26), the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) enhances Ca2+-induced MLC20 phosphorylation in a Rho-dependent manner. HA-1077 (10 µM) totally inhibits GTPgamma S-induced enhancement of MLC20 phosphorylation (Fig. 2A), suggesting the involvement of Rho kinase in this process. The inhibition of GTPgamma S-induced enhancement of MLC20 phosphorylation is HA-1077 dose dependent, with an IC50 value of ~2 µM (Fig. 2B). Importantly, the addition of HA-1077 up to 10 µM does not significantly inhibit Ca2+-induced MLC20 phosphorylation (Fig. 2, A and B). HA-1077 at 30 µM partially inhibits Ca2+-induced MLC20 phosphorylation. These observations indicate that HA-1077 at the effective concentrations does not inhibit MLCK in SMCs, which is consistent with the results shown in Table 1.


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Fig. 2.   HA-1077 inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation in permeabilized smooth muscle cells (SMCs). A: Ca2+-MLC20 phosphorylation relationship in the presence of Ca2+ alone, Ca2+ + HA-1077 (10 µM), Ca2+ + GTPgamma S (30 µM), and Ca2+ + GTPgamma S and HA-1077. B: dose-dependent inhibition by HA-1077 of MLC20 phosphorylation in the presence of Ca2+ (0.3 µM) alone and Ca2+ + GTPgamma S (30 µM). Each value represents the mean ± SE of 3 determinations.

We next studied the mechanism for Rho kinase-mediated enhancement of MLC20 phosphorylation by using HA-1077; we examined whether Rho kinase mediates inhibition of MLC20 dephosphorylation or potentiation of MLC20 phosphorylation in permeabilized SMCs. As we reported previously (26), GTPgamma S has a profound inhibitory effect on the dephosphorylation of MLC20; although MLC20 is gradually dephosphorylated over 10 min in the absence of GTPgamma S, in the presence of GTPgamma S the extent of MLC20 phosphorylation initially declines but stops decreasing at a level of ~0.45 at 5 min (Fig. 3A). In contrast, in the presence of GTPgamma S plus HA-1077 (10 µM), the MLC20 phosphorylation level falls down almost to zero within 5 min. Thus HA-1077 abolishes the inhibitory effect of GTPgamma S on MLC20 dephosphorylation. When adenosine 5'-O-(3-thiotriphosphate) is used as substrate instead of ATP, MLC20 is thiophosphorylated. Ca2+-induced thiophosphorylation of MLC20 continues to increase for up to 10 min (Fig. 3B), since thiophosphorylated MLC20 is resistant to the action of phosphatase (5). GTPgamma S did not enhance thiophosphorylation of MLC20 at any time point examined. Further addition of HA-1077 does not affect the levels of MLC20 thiophosphorylation. Thus HA-1077 reduces the extent of MLC20 phosphorylation by accelerating dephosphorylation of MLC20.


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Fig. 3.   A: HA-1077 prevents GTPgamma S-induced inhibition of MLC20 dephosphorylation. Permeabilized SMCs were first incubated in the phosphorylation buffer (0.3 µM Ca2+) with or without GTPgamma S (30 µM) and/or HA-1077 (10 µM) for 15 min and then were switched to the dephosphorylation buffer (the Ca2+-free, 2 mM EGTA, and 50 µM wortmannin-containing phosphorylation buffer) with or without GTPgamma S and/or HA-1077. Each value represents the mean ± SE of 3 determinations. B: neither GTPgamma S nor HA-1077 has any effect on thiophosphorylation of MLC20. Permeabilized SMCs were incubated in Ca2+ (0.1 µM) alone, Ca2+ + GTPgamma S (30 µM), or Ca2+ + GTPgamma S and HA-1077 (10 µM) in the presence of adenosine 5'-O-(3-thiotriphosphate) for the indicated time periods.

The above experiments were conducted using passaged SMCs derived from pig aorta. We examined whether the Rho kinase inhibitor HA-1077 inhibited sensitization of MLC20 phosphorylation in freshly isolated primary SMCs, as it does in passaged SMCs. The myosin content and expression of Rho A, Rho kinase, MBS, and PP1delta proteins are shown in Fig. 4A. Quantitation of densities of the proteins by densitometry shows that the amounts of the proteins in passaged SMCs are slightly smaller (78-96% of those in primary SMCs; Table 2). As shown in Fig. 4B, GTPgamma S enhanced Ca2+-induced MLC20 phosphorylation in both permeabilized primary SMCs and passaged SMCs. HA-1077 (10 µM) completely abolishes GTPgamma S enhancement of MLC20 phosphorylation in both cell types.


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Fig. 4.   Expression of the components of the Rho signaling pathways and inhibition by HA-1077 of GTPgamma S enhancement of Ca2+-induced MLC20 phosphorylation in freshly isolated primary vascular SMCs and passaged (passage 5) SMCs. A: cells were solubilized in Laemmli's SDS-sample buffer, and the protein concentrations were determined. Proteins (25 µg per lane) were separated on a 12% SDS-PAGE, followed by Coomassie blue staining (for myosin) and immunoblotting using respective specific antibodies [Rho A, Rho kinase, myosin-binding regulatory subunit (MBS), and PP1delta ]. Arrowheads indicate the bands corresponding to the respective antibodies. These bands were quenched by the inclusion of antigen peptides or proteins in the primary antibody solution of Western blotting. Nos. on left ordinate indicate the molecular masses of markers in kDa. MHC, myosin heavy chain. B: cells were permeabilized and treated as indicated. MLC20 phosphorylation was determined. Each value represents the mean ± SE of 3 determinations.


                              
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Table 2.   Expression levels of myosin heavy chains, Rho A, Rho kinase, MBS, and PP1delta

We also determined the effect of HA-1077 on intact vascular SMCs. Stimulation of intact SMCs with PGF2alpha (30 µM) induced time-dependent increases in both the monophosphorylated and diphosphorylated forms of MLC20 (Fig. 5). Treatment of SMCs with HA-1077 (10 µM) lowered the resting level of the monophosphorylated form of MLC20 and partially inhibited PGF2alpha -induced increases in monophosphorylated MLC20. HA-1077 exerted a profound inhibitory effect on levels of the diphosphorylated form of MLC20, totally abolishing the PGF2alpha -induced increase.


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Fig. 5.   HA-1077 inhibits PGF2alpha -induced increases in MLC20 phosphorylation in intact SMCs. Cells were pretreated or were not treated with HA-1077 (10 µM) for 10 min and then were stimulated with PGF2alpha (30 µM) for the indicated time periods. Levels of monophosphorylated (MLC-P) and diphosphorylated (MLC-P2) forms of MLC20 were determined. Values are means ± SE of results from 3 independent experiments.

We then determined how HA-1077 affects the myosin phosphatase activity of SMCs. Western blot analysis of the anti-MBS immunoprecipitate revealed the presence of 130-, 38-, and 21-kDa proteins, which were reactive with anti-MBS antibody, anti-PP1delta antibody, and anti-M21 antibody, respectively (Fig. 6A). We measured the phosphatase activity associated with the anti-MBS immunoprecipitate obtained from permeabilized SMCs. The amounts of immunoprecipitated MBS from the cells treated variously are shown in Fig. 6B. GTPgamma S stimulation of SMCs causes a 55% decrease in the phosphatase activity toward MLC20, and pretreatment of SMCs with C3 toxin abolishes this decrease (Fig. 6C). The addition of HA-1077 to cells reversed the GTPgamma S inhibition of the phosphatase activity dose dependently. These results are consistent with the notion that GTPgamma S-induced inhibition of myosin phosphatase is mediated through Rho and Rho kinase.


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Fig. 6.   HA-1077 and C3 prevents GTPgamma S-induced inhibition of myosin phosphatase activity. A: Western blot analysis of anti-MBS immunoprecipitate with anti-MBS, anti-PP1delta isoform, and anti-21-kDa regulatory subunit (M21) antibodies and Coomassie blue staining of anti-MBS immunoprecipitate. Nos. on left indicate molecular masses of marker proteins in kDa. Arrowheads indicate the position of a band specifically reactive to each antibody, which is quenched by the inclusion of antigen peptides or proteins in the primary antibody solution of Western blotting. Bands representing IgG and its degradation products are represented by . Coomassie staining shows the bands of IgG. B: anti-MBS immunoblots of anti-MBS immunoprecipitates. C: phosphatase activity associated with the anti-MBS immunoprecipitates. B and C: permeabilized SMCs were left unpretreated or were pretreated with C3 (1 µg/ml) or HA-1077 (3 or 10 µM) for 20 min and then were stimulated with GTPgamma S (30 µM) for 10 min. Cells were lysed, and myosin phosphatase was immunoprecipitated using anti-MBS antibody. Phosphatase activity associated with the immunoprecipitate was measured using 32P-labeled MLC20 as a substrate. Each value represents the mean ± SE of 3 determinations. **P < 0.01 by Dunnett's test compared with no stimulation; dagger  P < 0.05 and dagger dagger P < 0.01 compared with GTPgamma S stimulation in the absence of HA-1077 or C3.

We examined the effect of HA-1077 on the phosphorylation state of MBS in SMCs under the same conditions as described in Fig. 6, B and C. In permeabilized SMCs, the addition of GTPgamma S induced a threefold increase in the extent of phosphorylation of MBS that was nearly totally abolished by pretreatment with C3 toxin (Fig. 7). The addition of HA-1077 (10 µM) to cells also totally abolished the stimulatory effect of GTPgamma S. We further examined the involvement of Rho kinase in GTPgamma S-induced myosin phosphatase inhibition by studying the effect of the PH/cystein-rich domain of Rho kinase, a dominant inhibitor for Rho kinase (2). The addition of the recombinant PH/cystein-rich domain of Rho kinase (3 µM) to permeabilized SMCs inhibited GTPgamma S-induced enhancement of MLC20 phosphorylation by 75% (Fig. 8A), whereas the PH/cystein-rich domain of Rho kinase at 3 µM had no effect on MLC20 phosphorylation induced by Ca2+ alone. The addition of the Rho kinase PH/cystein-rich domain dose dependently inhibited GTPgamma S-induced MBS phosphorylation, with the complete inhibition obtained at 5 µM (Fig. 8B).


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Fig. 7.   HA-1077 and C3 inhibit GTPgamma S-induced phosphorylation of the 130-kDa MBS. Permeabilized SMCs were left unpretreated or were pretreated with HA-1077 (10 µM) for 10 min or C3 (1 µg/ml) for 20 min and then were stimulated with GTPgamma S (30 µM) for 10 min in the presence of [gamma -32P]ATP. MBS was immunoprecipitated and separated on 8% SDS-PAGE, followed by autoradiography. Autoradiographs showing 32P phosphorylation of MBS and anti-MBS blots are displayed on top. Radioactivity of the band corresponding to the MBS was determined by a Fuji BAS 2000 Bio-Image Analyzer and was expressed as the degree of increase over the radioactivity of a nonstimulated control. Each value represents the mean ± SE of 4 determinations. ** and dagger dagger P < 0.01 (by Dunnett's test) compared with no stimulation and GTPgamma S stimulation without pretreatment, respectively.



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Fig. 8.   Pleckstrin homology (PH)/cystein-rich domain of Rho kinase (PH domain) inhibits GTPgamma S-induced enhancement of MLC20 phosphorylation and MBS phosphorylation in permeabilized SMCs. A: cells were incubated with Ca2+ (0.3 µM) alone or Ca2+ + GTPgamma S (30 µM) in the presence of various concentrations of the recombinant PH domain of Rho kinase. B: permeabilized SMCs were incubated with the recombinant PH/cystein-rich domain at the indicated concentrations for 10 min and then were stimulated with GTPgamma S (30 µM) for a further 10 min in the presence of [gamma -32P]ATP. Phosphorylation of MBS was analyzed as described in the legend for Fig. 7. Each value represents the mean ± SE of 3 determinations. **P < 0.01 by Dunnett's test compared with no stimulation; dagger P < 0.05 and dagger dagger P < 0.01 compared with GTPgamma S stimulation in the absence of PH domain. Representative autoradiographs and anti-MBS Western blots are shown on top in B.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Accumulating evidence shows that Rho kinase is one of the major Rho effectors that are implicated in the regulation of various cell functions (2, 20, 25, 34). The reported substrate proteins for Rho kinase include MBS (15), MLC20 (3), vimentin and glial fibrillary acidic protein (17), the ezrin/radixin/moesin family proteins (23), and adducin (25, 34). A specific Rho kinase inhibitor would be beneficial to see how Rho kinase-catalyzed phosphorylation of these proteins affects cell functions. In the present study, we demonstrated that HA-1077 acts as a potent in vivo Rho kinase inhibitor. We next analyzed a role for Rho kinase in Rho-dependent myosin phosphatase inhibition in vascular SMCs by using this compound, demonstrating that in vivo Rho kinase acts downstream of Rho to induce phosphorylation of MBS, resulting in inhibition of myosin phosphatase and consequent enhancement of MLC20 phosphorylation.

It was recently shown that treatment of intact platelets with a thromboxane A2 analog induced a decrease in the activity of myosin phosphatase isolated by immunoprecipitation using anti-MBS antibody (24). The association of Rho and Rho kinase with anti-MBS immunoprecipitate was observed; however, no functional involvement of Rho and Rho kinase in the agonist-induced myosin phosphatase inhibition was shown (24). It was also recently shown in vascular endothelial cells that thrombin inhibited phosphatase activity toward MLC20 in myosin-enriched cell fractions in a Rho-dependent manner (6). However, it was not directly demonstrated that Rho kinase mediated the thrombin-induced inhibition of phosphatase activity. Also, the effect of thrombin on the phosphorylation status of MBS was not shown (6). In the present study, we demonstrated that GTPgamma S stimulation indeed leads to inhibition of myosin phosphatase activity in a Rho-dependent manner in SMCs (Fig. 6). We further found that the Rho kinase inhibitor HA-1077 totally abolishes GTPgamma S inhibition of myosin phosphatase activity and consequent enhancement of MLC20 phosphorylation (Figs. 2, 3, and 6). In agreement with this, HA-1077 strongly inhibits the receptor agonist-induced increase in phosphorylation, especially diphosphorylation, of MLC20 in intact SMCs (Fig. 5). The observations indicate that, among Rho targets, Rho kinase is responsible for mediating myosin phosphatase suppression in smooth muscle. Moreover, the present study shows that GTPgamma S-induced, Rho-dependent myosin phosphatase inhibition is accompanied by a concomitant increase in phosphorylation of MBS, which is also blocked by HA-1077 (Fig. 7). HA-1077 exhibits an inhibitor activity for another Rho-associated protein kinase, PKN, as well (Table 1; see Refs. 2 and 18). However, it was demonstrated that PKN, in vitro, neither phosphorylated myosin phosphatase nor inhibited its activity (13). These observations are thus consistent with the notion that, in vivo in smooth muscle, Rho kinase mediates phosphorylation of MBS to result in the inhibition of phosphatase activity. The observations in Fig. 8 that the recombinant PH/cystein-rich domain of Rho kinase, a dominant inhibitor for Rho kinase, inhibited GTPgamma S-induced enhancement of MLC20 phosphorylation and MBS phosphorylation provides further support for the role of Rho kinase in the regulation of myosin phosphatase.

It was reported recently (18) that the addition of the constitutively active catalytic fragment of Rho kinase to permeabilized vascular smooth muscle preparations caused a Ca2+/calmodulin-independent phosphorylation of MLC20 and contraction. It was also demonstrated previously (3) that Rho kinase phosphorylates purified myosin in vitro at the MLCK phosphorylation site (Ser19) of MLC20 and increases actin-activated myosin ATPase activity. These observations may suggest that direct phosphorylation of MLC20 by Rho kinase could contribute to a Rho kinase-mediated increase in MLC20 phosphorylation under certain experimental conditions. However, we (26) and others (16) previously observed in permeabilized SMCs and vascular strips that GTPgamma S stimulation did not increase Ca2+-induced thiophosphorylation of MLC20. Because thiophosphorylated MLC20 is resistant to the action of myosin phosphatase (5), these observations indicate that the myosin kinase activity is not enhanced in GTPgamma S-stimulated smooth muscle. Furthermore, in the present study, the Rho kinase inhibitor HA-1077 does not inhibit Ca2+-induced thiophosphorylation of MLC20 in the presence of GTPgamma S but does reverse GTPgamma S-induced inhibition of MLC20 dephosphorylation (Fig. 3). These results indicate that in SMCs, at least under the present conditions, Rho-dependent enhancement of MLC20 phosphorylation is mediated largely through the downregulation of myosin phosphatase activity.

It was recently reported that a new compound, Y-27632 (33), which has a totally different molecular structure from HA-1077, potently inhibits Rho kinase and its isoform ROCKI/ROKbeta . The Ki value of Y-27632 for ROCKI was calculated to be 0.14 µM, which was based upon the measured Km value (0.1 µM) of ROCKI for ATP. Consistent with our results, Y-27632 was shown to inhibit GTPgamma S enhancement of Ca2+-induced smooth muscle contraction (7, 33). However, the effects of Y-27632 on MBS phosphorylation and myosin phosphatase activity were not reported. It was also shown that Y-27632 lowers blood pressure in rat hypertension models. The Rho kinase inhibitors would serve as useful tools for dissecting the roles for Rho kinase in the regulation of functions of smooth muscle and nonmuscle cells.


    ACKNOWLEDGEMENTS

We thank Dr. C. Fukazawa for help in raising antibodies and R. Suzuki and N. Yamaguchi for secretarial assistance.


    FOOTNOTES

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and by the Japan Society for the Promotion of Science "Resarch for the Future" Program.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Y. Takuwa, Dept. of Physiology, Kanazawa Univ. School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan (E-mail: ytakuwa{at}med.kanazawa-u.ac.jp).

Received 11 February 1999; accepted in final form 19 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alessi, D., L. K. MacDougall, M. M. Sola, M. Ikebe, and P. Cohen. The control of protein phosphatase-1 by targeting subunits: the major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur. J. Biochem. 210: 1023-1035, 1992.

2.   Amano, M., K. Chihara, K. Kimura, Y. Fukata, N. Nakamura, Y. Matsuura, and K. Kaibuchi. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275: 1308-1311, 1997[Abstract/Free Full Text].

3.   Amano, M., M. Ito, K. Kimura, Y. Fukata, K. Chihara, T. Nakano, Y. Matsuura, and K. Kaibuchi. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271: 20246-20249, 1996[Abstract/Free Full Text].

4.   Asano, T., T. Suzuki, M. Tsuchiya, S. Satoh, I. Ikegaki, M. Shibuya, Y. Suzuki, and H. Hidaka. Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br. J. Pharmacol. 98: 1091-1100, 1989[Abstract].

5.   Eto, M., T. Ohmori, M. Suzuki, K. Furuya, and F. Morita. A noved protein phosphatase-1 inhibitory protein potentiated by protein kinase C Isolation from poruine aorta media and characterization. J. Biochem. (Tokyo) 118: 1104-1107, 1995[Abstract].

6.   Eyk, J. E., D. K. Arrell, D. B. Fosten, J. P. Strauss, T. Y. K. Heinonen, E. Furmaniak-Kazmierczak, G. P. Cote, and A. S. Mak. Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. J. Biol. Chem. 273: 23433-23439, 1998[Abstract/Free Full Text].

7.   Fu, X., M. C. Gong, T. Jia, A. V. Somlyo, and A. P. Somlyo. The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPgamma S-, and phorbor ester-induced Ca2+-sensitization of smooth muscle. FEBS Lett. 440: 183-187, 1998[ISI][Medline].

8.   Fujita, A., T. Takeuchi, H. Nakajima, H. Nishio, and F. Hata. Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C. J. Pharmacol. Exp. Ther. 274: 555-561, 1995[Abstract].

9.   Gong, M. C., K. Iizuka, G. Nixon, J. P. Browne, A. Hall, J. F. Eccleston, M. Sugai, S. Kobayashi, A. V. Somlyo, and A. P. Somlyo. Role of guanine nucleotide-binding proteins-ras-family or trimeric proteins or both in Ca2+ sensitization iof smooth muscle. Proc. Natl. Acad. Sci. USA 93: 1340-1345, 1996[Abstract/Free Full Text].

10.   Hall, A. Small GTP-binding proteins and the regulation of the actin cytoskelton. Annu. Rev. Cell Biol. 10: 31-54, 1994[ISI].

11.   Hirata, K., A. Kikuchi, S. Sasaki, S. Kuroda, K. Kaibuchi, Y. Matsuura, H. Seki, K. Saida, and Y. Takai. Involvement of rho p21 in the GTPgamma S-enhanced calcium ion sensitivity of smooth muscle contraction. J. Biol. Chem. 267: 8719-8722, 1992[Abstract/Free Full Text].

12.   Hoar, P. E, and W. G. L. Kerrick. Chicken gizzard: relation between calcium-activated phosphorylation and contraction. Science 204: 503-506, 1979[ISI][Medline].

13.   Ishizaki, T., M. Maekawa, K. Fujisawa, K. Okawa, A. Iwamatsu, A. Fujita, N. Watanabe, T. Saito, A. Kakizuka, N. Morii, and S. Narumiya. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonia dystrophy kinase. EMBO J. 15: 1885-1893, 1996[Abstract].

14.   Kamm, K. E., and J. T. Stull. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu. Rev. Pharmacol. Toxicol. 25: 593-620, 1985[ISI][Medline].

15.   Kimura, K., M. Ito, M. Amano, K. Chihara, Y. Fukata, M. Nakafuku, B. Yamamori, J. Feng, T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996[Abstract].

16.   Kitazawa, T., M. Masuo, and A. P. Somlyo. Protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc. Natl. Acad. Sci. USA 88: 9307-9310, 1991[Abstract].

17.   Kosako, H., M. Amano, M. Yanagida, K. Tanabe, Y. Nishi, K. Kibuchi, and M. Inagaki. Phosphorylation of glial fibrillar acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase. J. Biol. Chem. 272: 10333-10336, 1997[Abstract/Free Full Text].

18.   Kureishi, Y., S. Kobayashi, M. Amano, K. Kimura, H. Kanaide, T. Nakano, K. Kaibuchi, and M. Ito. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J. Biol. Chem. 272: 12257-12260, 1997[Abstract/Free Full Text].

19.   Leung, T., X. Q. Chen, E. Manser, and L. Lim. The p160 RhoA-binding kinase ROKalpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16: 5313-5327, 1996[Abstract].

20.   Lim, L., E. Manser, T. Leung, and C. Hall. Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur. J. Biochem. 242: 171-185, 1996[Abstract].

21.   Lucius, C., A. Anders, A. Steusloff, M. Troschka, F. Hofman, K. Aktories, and G. Pfitzer. Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins. J. Physiol. (Lond.) 506: 83-93, 1998[Abstract/Free Full Text].

22.   Matsui, T., M. Amano, T. Yamamoto, K. Chihara, M. Nakafuku, M. Ito, T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for the small GTP-binding protein Rho. EMBO J. 15: 2208-2216, 1996[Abstract].

23.   Matsui, T., M. Maeda, Y. Doi, S. Yanemura, M. Amano, K. Kaibuchi, S. Tsukita, and S. Tsukita. Rho-kinase phosphorylates COOH-terminal threonines of exrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140: 647-657, 1998[Abstract/Free Full Text].

24.   Nakai, K., Y. Suzuki, H. Kihara, H. Wada, M. Fujioka, M. Ito, T. Nakana, K. Kaibuchi, H. Shiku, and M. Nishikawa. Regulation of myosin phosphatase through phosphorylation of the myosin-binding subunit in platelet activation. Blood 90: 3936-3942, 1997[Abstract/Free Full Text].

25.   Narumiya, S., T. Ishizaki, and N. Watanabe. Rho effectoors and reorganization of actin cytoskeleton. FEBS Lett. 410: 68-72, 1997[ISI][Medline].

26.   Noda, M., C. Yasuda-Fukazawa, K. Moriishi, T. Kato, T. Okuda, K. Kurokawa, and Y. Takuwa. Involvement of rho in GTPgamma S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Lett. 367: 246-250, 1995[ISI][Medline].

27.   Seto, M., Y. Sasaki, H. Hidada, and Y. Sasaki. Effects of HA1077, a protein kinase inhibitor, on myosin phosphorylation and tension in smooth muscle. Eur. J. Pharmacol. 195: 267-272, 1991[ISI][Medline].

28.   Shimizu, H., M. Ito, M. Miyahara, K. Ichikawa, S. Okubo, T. Konishi, M. Naka, T. Tanaka, K. Hirano, D. J. Hartshorne, and T. Nakano. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J. Biol. Chem. 269: 30407-30411, 1994[Abstract/Free Full Text].

29.   Takai, Y., T. Sasaki, K. Tanaka, and H. Nakanishi. Rho as a regulator of the cytoskeleton. Trends Biochem. Sci. 20: 227-231, 1995[ISI][Medline].

30.   Takuwa, Y. Regulation of vascular smooth muscle contraction. The roles of Ca2+, protein kinase C and myosin light chain phosphatase. Jpn. Heart J. 37: 793-813, 1996[ISI][Medline].

31.   Takuwa, N., W. Zhou, M. Kumada, and Y. Takuwa. Ca2+-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. J. Biol. Chem. 268: 138-145, 1993[Abstract/Free Full Text].

32.   Tan, J. L., S. Ravid, and J. A. Spudich. Control of nonmuscle myosin by phosphorylation. Annu. Rev. Biochem. 61: 721-759, 1992[ISI][Medline].

33.   Uehata, M., T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita, H. Tamakawa, K. Yamagami, J. Inui, M. Maekawa, and S. Narumiya. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997[ISI][Medline].

34.   Van Aelst, L., and C. D'Souza-Schorey. Rho GTPases and signaling networks. Genes Dev. 11: 2295-2322, 1997[Free Full Text].


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