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
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ABSTRACT |
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)
(GTP
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 GTP
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
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INTRODUCTION |
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/ROK
/ROCKII (19, 22) and its close relative ROK
/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 1
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.
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MATERIALS AND METHODS |
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
-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
[
-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-PP1
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 PP1
, 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
-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
[
-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 DH5
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 |
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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We then examined how HA-1077 affected the Rho-mediated regulation of
MLC20 phosphorylation in vascular
SMCs (Fig. 2). In
-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)
(GTP
S) enhances Ca2+-induced
MLC20 phosphorylation in a
Rho-dependent manner. HA-1077 (10 µM) totally inhibits
GTP
S-induced enhancement of
MLC20 phosphorylation (Fig.
2A), suggesting the involvement of
Rho kinase in this process. The inhibition of GTP
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)
(GTP 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+ + GTP S
(30 µM), and
Ca2+ + GTP 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+ + GTP S (30 µM). Each value represents the mean ± SE of 3 determinations.
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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),
GTP
S has a profound inhibitory effect on the dephosphorylation of
MLC20; although
MLC20 is gradually
dephosphorylated over 10 min in the absence of GTP
S, in the presence
of GTP
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 GTP
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 GTP
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). GTP
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 GTP S-induced
inhibition of MLC20
dephosphorylation. Permeabilized SMCs were first incubated in the
phosphorylation buffer (0.3 µM
Ca2+) with or without GTP 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 GTP S
and/or HA-1077. Each value represents the mean ± SE of 3 determinations. B: neither GTP S nor
HA-1077 has any effect on thiophosphorylation of
MLC20. Permeabilized SMCs were
incubated in Ca2+ (0.1 µM)
alone, Ca2+ + GTP S
(30 µM), or
Ca2+ + GTP S and
HA-1077 (10 µM) in the presence of adenosine
5'-O-(3-thiotriphosphate) for
the indicated time periods.
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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 PP1
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, GTP
S enhanced
Ca2+-induced
MLC20 phosphorylation in both
permeabilized primary SMCs and passaged SMCs. HA-1077 (10 µM)
completely abolishes GTP
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 GTP 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
PP1 ]. 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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We also determined the effect of HA-1077 on intact vascular SMCs.
Stimulation of intact SMCs with
PGF2
(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
PGF2
-induced increases in
monophosphorylated MLC20. HA-1077
exerted a profound inhibitory effect on levels of the diphosphorylated
form of MLC20, totally abolishing
the PGF2
-induced increase.

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Fig. 5.
HA-1077 inhibits PGF2 -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
PGF2 (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.
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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-PP1
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. GTP
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 GTP
S inhibition of the phosphatase activity dose
dependently. These results are consistent with the notion that
GTP
S-induced inhibition of myosin phosphatase is mediated through
Rho and Rho kinase.

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Fig. 6.
HA-1077 and C3 prevents GTP S-induced inhibition of myosin
phosphatase activity. A: Western blot
analysis of anti-MBS immunoprecipitate with anti-MBS, anti-PP1
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 GTP 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;
P < 0.05 and
 P < 0.01 compared with GTP S stimulation in the
absence of HA-1077 or C3.
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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 GTP
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 GTP
S. We further examined the involvement of
Rho kinase in GTP
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 GTP
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 GTP
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 GTP 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 GTP S (30 µM) for 10 min in the presence of
[ -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
 P < 0.01 (by
Dunnett's test) compared with no stimulation and GTP S stimulation
without pretreatment, respectively.
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Fig. 8.
Pleckstrin homology (PH)/cystein-rich domain of Rho kinase (PH domain)
inhibits GTP S-induced enhancement of
MLC20 phosphorylation and MBS
phosphorylation in permeabilized SMCs.
A: cells were incubated with
Ca2+ (0.3 µM) alone or
Ca2+ + GTP 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
GTP S (30 µM) for a further 10 min in the presence of
[ -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;
P < 0.05 and  P < 0.01 compared with GTP S stimulation in the absence of PH domain.
Representative autoradiographs and anti-MBS Western blots are shown on
top in
B.
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 |
DISCUSSION |
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 GTP
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 GTP
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 GTP
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 GTP
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
GTP
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
GTP
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
GTP
S but does reverse GTP
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/ROK
. 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 GTP
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.
 |
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