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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 |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 GTPS-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 |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 -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.
-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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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)
(GTPS) enhances Ca2+-induced
MLC20 phosphorylation in a
Rho-dependent manner. HA-1077 (10 µM) totally inhibits
GTPS-induced enhancement of
MLC20 phosphorylation (Fig.
2A), suggesting the involvement of
Rho kinase in this process. The inhibition of GTPS-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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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),
GTPS has a profound inhibitory effect on the dephosphorylation of
MLC20; although
MLC20 is gradually
dephosphorylated over 10 min in the absence of GTPS, in the presence
of GTPS 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 GTPS 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 GTPS 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). GTPS 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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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, GTPS enhanced
Ca2+-induced
MLC20 phosphorylation in both
permeabilized primary SMCs and passaged SMCs. HA-1077 (10 µM)
completely abolishes GTPS enhancement of
MLC20 phosphorylation in both cell
types.
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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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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. GTPS 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 GTPS inhibition of the phosphatase activity dose
dependently. These results are consistent with the notion that
GTPS-induced inhibition of myosin phosphatase is mediated through
Rho and Rho kinase.
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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 GTPS 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 GTPS. We further examined the involvement of
Rho kinase in GTPS-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 GTPS-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 GTPS-induced MBS phosphorylation, with the complete inhibition obtained at 5 µM (Fig.
8B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 GTPS 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 GTPS 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 GTPS-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 GTPS-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
GTPS 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
GTPS-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
GTPS but does reverse GTPS-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 GTPS 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.
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ACKNOWLEDGEMENTS |
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We thank Dr. C. Fukazawa for help in raising antibodies and R. Suzuki and N. Yamaguchi for secretarial assistance.
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FOOTNOTES |
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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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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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. 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
P < 0.05 and
