Molecular Medicine |
From the Department of Neurosurgery, Molecular Biology Research Laboratory, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, and the Division of Signal Transduction (K.K.), Nara Institute of Science and Technology, Ikoma, Nara, Japan
| Abstract |
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Key Words: vasospasm Rho-kinase myosin light chain phosphorylation dogs
| Introduction |
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-Sinduced increase in the force/Ca2+
ratio, so-called Ca2+
sensitization.9 10 11 12 13 14 Therefore, an additional mechanism
that can regulate Ca2+ sensitization of vascular
smooth muscle has been considered. With the use of membrane
permeabilization of smooth muscle, the possibility that monomeric Ras
family G proteins, such as Rho, contribute to
Ca2+ sensitization of smooth muscle was
demonstrated.13 15 16 In permeabilized
smooth muscle cells, GTP-
-S induces MLC
phosphorylation at submaximal
Ca2+ concentration by inhibiting the
dephosphorylation of MLC,10 13 presumably
by activating Rho.17 Recently, Rho-kinase, which is
activated by Rho,16 18 19 has been reported to
phosphorylate not only MLC stoichiometrically at the site
that is phosphorylated by
Ca2+/CaM-dependent MLC kinase20 but
also myosin phosphatase at its myosin-binding subunit (MBS), thus
inactivating it in vitro.21 These findings in a cell-free
system, plus the reports of G-proteinmediated
Ca2+ sensitization of smooth muscle
contraction,13 14 22 23 suggest that Rho-kinase may induce
contraction and concomitant MLC phosphorylation of the
smooth muscle.24 25 In the present study, we examine
whether Rho-kinase is involved in the development of cerebral vasospasm
in the presence of elevated intracellular Ca2+
levels. | Materials and Methods |
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The phosphorylations of MLC and MBS in myosin phosphatase and the activity of Rho-kinase were studied. The phosphorylation of MLC was examined by urea-glycerol gel electrophoresis and immunoblot analysis with anti-MLC antibody, and phosphorylated MLC was expressed as a percentage of total MLC (ie, unphosphorylated MLC plus phosphorylated MLC). Rho-kinase activity was examined by Rho-kinase assay and immunoblot analysis with antiglutathione-S-transferase-bovine Rho-kinase antibody and calculated by dividing the densimetric values of Rho-kinase activity by those of the immunoreactive Rho-kinase level. The phosphorylation of MBS was probed separately with anti-pS854 antibody, which was prepared against the synthetic phosphopeptide pS854 (CREKRR phosphoS854TGVSF) as an antigen, and with antiglutathione-S-transferase-MBS-COOH terminal antibody and calculated by dividing the densitometric values of the phosphorylation levels of MBS by those of the immunoreactive levels of MBS. Finally, after the basilar artery in vasospasm on day 7 was exposed transclivally, it was treated by a topical 30-minute application of successively increasing concentrations of Y-27632, a specific Rho-kinase inhibitor. At the end of experiment, 10 mmol/L EGTA was added, and the reduced diameter of the spastic basilar artery was expressed as a percentage of the relaxation induced by EGTA.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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45 minutes after treatment and then
stabilized, as shown in Figure 2
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Phosphorylation of MLC and Activation of Rho-Kinase
Associated With Phosphorylation of MBS
Previously, we confirmed that MLC in the spastic basilar artery
was phosphorylated by
Ca2+/CaM-dependent MLC kinase but not by protein
kinase C.5 6 Therefore, we examined by glycerol-urea gel
electrophoresis and subsequent immunoblot analysis
whether the phosphorylation of MLC was increased in
vasospasm. As shown in Figure 3
, the
monophosphorylation of MLC was increased with the
passage of time in vasospasm. In addition, the Rho-kinase assay
demonstrated a progressive increase in Rho-kinase activity in the
basilar artery in vasospasm on days 0, 2, and 7, as shown in Figure 4A
, but immunoreactive Rho-kinase levels
were not changed (Figure 4B
), demonstrating a significant
increase in Rho-kinase activity in vasospasm with the lapse of time
after SAH (Figure 4E
). The Rho-kinase activities were increased
slightly in the KCl-15 subgroup, moderately in the KCl-90 and
serotonin-15 subgroups, and markedly in the
serotonin-90 subgroup, as shown in Figure 4F
. Thus,
the Rho-kinase activities in vasospasm on days 0 and 2 were comparable
to those in the KCl-90 or serotonin-15 subgroup and in the
serotonin-90 subgroup, respectively. One of the major sites
of phosphorylation of MBS by Rho-kinase has been
identified as Ser854, and the antibody that specifically recognizes MBS
phosphorylated at Ser854 has been
developed.26 When MBS phosphorylation of
myosin phosphatase was immunologically examined with use of an
anti-pS854 antibody, the phosphorylation of MBS was
shown to increase in vasospasm on days 0 and 2 and particularly on day
7 (Figure 4C
), without any significant changes in the
immunoreactive MBS level (Figure 4D
), demonstrating a
significant increase in MBS phosphorylation in
vasospasm, particularly on day 7 (Figure 4E
).
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Dilation of Spastic Artery by Y-27632
Representative angiograms of the basilar artery in
the spastic group on day 7 are shown in Figure 5
. The mean±SEM diameter of the spastic
basilar arteries on day 7 was reduced to 56±3% of the control caliber
(Figure 1
). To define the involvement of Rho-kinase in the
development of vasospasm, Y-27632, a specific inhibitor of
Rho-kinase,27 was topically applied to the exposed spastic
basilar artery on day 7 to examine the responses of caliber as well as
the phosphorylation of MBS and of MLC in the spastic
basilar artery. Before the topical application of Y-27632, the blood
clot around the exposed spastic basilar artery was carefully removed to
avoid inducing significant changes in the caliber of the spastic
artery. The exposed spastic basilar artery was dilated dose-dependently
by a topical application of
3x10-7 to
1x10-4 mol/L Y-27632, as
shown in Figure 5
and the Table
. Taking
the relaxation induced by 10 mmol/L EGTA as 100%, the mean±SEM
ED50 value of Y-27632 in 3 dogs was 2.25±
0.28 µmol/L ( Table
). The topical application of 10
µmol/L Y-27632 to the spastic basilar artery on day 7 in 3 dogs
induced 69.7±2.6% relaxation, as shown in the Table
. In
addition, MBS phosphorylation levels were decreased
from 870±73.5% to 105±6.2% (Figures 4C
and 4E
), and MLC
phosphorylation levels were decreased from 74.4±9.5%
to 22.8±8.2% (Figure 6
). Thus, the
extent of decrease in MLC phosphorylation after the
topical application of 10 µmol/L Y-27632 (Figure 6
) was
consistent with that of dilatation of the exposed spastic
basilar artery ( Table
). The topical application of 10
mmol/L EGTA reduced MLC phosphorylation in vasospasm on
day 7 from 74.4±9.5% to 0.5±0.2%, as shown in Figure 6
.
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| Discussion |
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-S induces minimal MLC
phosphorylation in permeabilized smooth
muscle in the presence of EGTA.10 15 Because the level of
MBS phosphorylation in vasospasm on day 7 is much
higher than that the level on day 2, Rho-kinase may play more critical
roles at the later stage in vasospasm.
The topical application of 10 µmol/L Y-27632 to the spastic
basilar artery on day 7 induced a concurrent decrease in contraction
and phosphorylation of MBS and MLC. Y-27632 is a
specific inhibitor of Rho-kinase and selectively inhibits
agonist (including serotonin)induced smooth muscle
contraction by inhibiting Ca2+ sensitization
(Ki 0.14 for Rho-kinase,
Ki 26 for protein kinase C, and
Ki >250 for MLC
kinase).27 The concentration of Y-27632 used in the
present study inhibits GTP-
-Sinduced contraction of
permeabilized strips of rabbit mesentery
artery by
80% and phenylephrine-induced contraction
of rabbit aortic strips by
90% but has little effect on
Ca2+-induced and calyculin Ainduced contraction
of permeabilized strips of rabbit mesentery
artery.27 Rho-kinase inhibition by Y-27632 in vasospasm
has demonstrated that MBS phosphorylation is more
decreased than is MLC phosphorylation and that the MLC
phosphorylation induced by
Ca2+/CaM-dependent MLC kinase in vasospasm
because of the continuous elevation of intracellular
Ca2+ levels2 3 may not be affected
by Y-27632. Therefore, the Y-27632specific MLC
phosphorylation in vasospasm may be mediated by
Rho-kinase mainly through the inhibition of myosin
phosphatase.21 Thus, although the intracellular
Ca2+ level is elevated in
vasospasm,2 3 the activation of Rho-kinase associated with
the elevation of MBS phosphorylation and the
Y-27632sensitive MLC phosphorylation in the spastic
basilar artery suggests the involvement of Rho-kinase in the
enhancement of vasospasm, namely, a Ca2+
sensitization mechanism.9 14 21 23 30
The present study (together with the previous studies) indicates
that the RhoRho-kinase pathway is activated during the
development of cerebral vasospasm. How is the RhoRho-kinase pathway
activated? Several recent studies imply that some trimeric
G-proteincoupled receptors, including lysophosphatidic acid,
thrombin, and serotonin receptors, are linked to the
RhoRho-kinase pathway.31 The
subunits from
Gi, Gq,
G12, and G13 are postulated
to activate Rho by regulating GDP/GTP exchange factors (GEFs)
for Rho.32 33 34 35 For
G
13, a direct
interaction with a specific RhoGEF, p115-RhoGEF, is known to enhance
exchange activity and Rho-GTP binding.34 35 Although the
mechanism by which SAH activates the RhoRho-kinase pathway
remains to be clarified, it is tempting to speculate that certain
ligands for the G-proteincoupled receptors, which are produced after
SAH, activate Rho (and subsequently Rho-kinase) and enhance
cerebral vasospasm. The activation of the tyrosine kinase pathway in
vasospasm6 suggests an involvement of G-proteincoupled
receptors in addition to receptor protein tyrosine kinase. The
monophosphorylation of MLC in the spastic basilar
artery on day 7 was almost completely inhibited by the topical
application of 10 mmol/L EGTA. Because the concentration of EGTA
used in the present study induces cytosolic
Ca2+ levels to nominally zero,31
Ca2+/CaM-dependent MLC kinase in the spastic
basilar artery on day 7 could be almost completely
inactivated when EGTA was applied topically. In
contrast, because Rho-kinase is theoretically insensitive to 10
mmol/L EGTA, Rho-kinase in the spastic basilar artery on day 7, after
topical application of EGTA, would be still capable of phosphorylating
not only MLC20 but also myosin phosphatase, thus
inactivating it.21 Therefore, the nominally zero
phosphorylation of MLC in the spastic basilar artery on
day 7 induced by the topical application of EGTA would be mediated
mainly by the complete inhibition of
Ca2+/CaM-dependent MLC kinase, although we could
not rule out the possibility that Rho-kinase activity is inhibited
under the conditions.
The intracellular Ca2+ levels in the canine basilar artery were transiently elevated only at the beginning of serotonin-induced contraction.2 3 Therefore, serotonin-induced contraction is mainly dependent on the activation of Rho-kinase, according to the evidence of marked relaxation of serotonin-induced smooth muscle contraction by Y-27632.27 In contrast, the intracellular Ca2+ levels in the spastic basilar artery are continuously increased according to the evidence of µ-calpain activation,2 3 and the MLC phosphorylation in the spastic basilar artery is reported to be mediated by Ca2+/CaM-dependent MLC kinase.5 6 However, the maximum response of the spastic basilar artery to ML-9, a selective inhibitor of Ca2+/CaM-dependent MLC kinase, was less than that to EGTA,5 suggesting that mechanisms other than the participation of Ca2+/CaM-dependent MLC kinase (eg, involvement of Rho-kinase) operate in vasospasm. Therefore, the present study suggests that Rho-kinase works synergistically with Ca2+/CaM-dependent MLC kinase in vasospasm and that the vascular smooth muscle cells need such a 2-pronged approach on MLC phosphorylation to sustain the uninterrupted increase in cellular contractility characteristic of vasospasm. It remains to be determined to what extent Rho-kinase and Ca2+/CaM-dependent MLC kinase are involved in the development of vasospasm and to detect other mechanisms responsible for the development of vasospasm. However, from the therapeutic point of view, it is noted that the topical application of EGTA to the spastic basilar artery demonstrates the maximal relaxation compared with other reagents, such as inhibitors of Rho-kinase or Ca2+/CaM-dependent MLC kinase.
| Acknowledgments |
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| Footnotes |
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Received September 20, 1999; accepted June 9, 2000.
| References |
|---|
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in
canine basilar artery in vasospasm. J Neurosurg. 1997;87:752756.[Medline]
[Order article via Infotrieve]
-adrenergic
Ca2+ release in smooth muscle:
physiological role of inositol 1,4,5-trisphosphate
in pharmacomechanical coupling. J Biol Chem. 1989;264:1799718004.
S-dependent regulation of smooth muscle contractile
elements. Am J Physiol. 1992;262:C405C410.
S-induced
enhancement of phosphorylation of 20 kDa myosin light
chain in vascular smooth muscle cells: inhibition of phosphatase
activity. FEBS Lett. 1995;367:246250.[Medline]
[Order article via Infotrieve]
-independent coupling of
2-adrenergic receptor to
p21rhoA in preadipocytes. J Biol
Chem. 1998;273:1580415810.
12,
G
13, and
G
q induce Rho-dependent
neurite retraction through different signaling pathways. J
Biol Chem. 1998;273:2870028707.
12 and
G
13.
Science. 1998;280:21092111.
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