© 2000 American Heart Association, Inc.
Molecular Medicine |
Involvement of Rho-Kinase–Mediated Phosphorylation of Myosin Light Chain in Enhancement of Cerebral Vasospasm
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
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Abstract—Subarachnoid hemorrhage (SAH) often induces a long-term narrowing of the cerebral artery called cerebral vasospasm. Myosin light chain (MLC) in the spastic basilar artery was reported previously to be phosphorylated by Ca2+/calmodulin-dependent MLC kinase. Because Rho-kinase, which is activated by the small GTPase Rho, phosphorylates not only MLC but also myosin phosphatase at its myosin-binding subunit (MBS), thus inactivating myosin phosphatase, we examined whether Rho-kinase is involved in the development of vasospasm. Cerebral vasospasm was produced in the canine basilar artery by a 2-hemorrhage method, and vasocontractions were induced by topical application of 80 mmol/L KCl or 0.5 µmol/L serotonin to the canine basilar artery exposed transclivally. The phosphorylation of MLC in the basilar artery was increased concurrently with an enhancement in the intensity of vasospasm with the passage of time after SAH. In addition, Rho-kinase in the basilar artery was activated concurrently with an increase in the phosphorylation of MBS at Ser854 in vasospasm. The Rho-kinase activation levels in vasospasm on days 0 and 2 were comparable to those in KCl- and serotonin-induced sustained vasocontraction, respectively, and those in vasospasm on day 7 were markedly high. The topical application of Y-27632, a specific inhibitor of Rho-kinase, to the exposed spastic basilar artery on day 7 induced a dose-dependent dilation, and the intensities of vasospasm and the phosphorylation of MBS and MLC were simultaneously decreased by 10 µmol/L Y-27632, although the decrease in MBS phosphorylation was more marked than the decrease in MLC phosphorylation. These results indicate that the activation of Rho-kinase and the phosphorylation of MLC and MBS occur concomitantly during vasospasm induced by SAH and suggest that Rho-kinase is involved in the enhancement of cerebral vasospasm in addition to Ca2+/calmodulin-dependent MLC kinase by increasing the phosphorylation of MLC directly or indirectly as a result of the inhibition of myosin phosphatase by its phosphorylation.
Key Words: vasospasm • Rho-kinase • myosin light chain • phosphorylation • dogs
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Introduction |
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The management of patients with acutely ruptured intracranial aneurysms should achieve 2 major goals: (1) prevention of subsequent bleeding and (2) prevention and treatment of cerebral vasospasm, which usually develops between 4 and 14 days after subarachnoid hemorrhage (SAH). Despite the improvements in management of aneurysmal SAH, vasospasm remains an important cause of the morbidity and mortality accompanying this disease.1 Recently, it has been reported that µ-calpain, but not m-calpain, in the canine basilar artery is continuously activated in vasospasm,2 3 indicating a continuous rise of intracellular Ca2+ levels, because µ- and m-calpains are activated at 1 µmol/L and 1 mmol/L Ca2+ levels, respectively.4 Myosin light chain (MLC) in the spastic canine basilar artery has been shown to be phosphorylated by Ca2+/calmodulin (CaM)-dependent MLC kinase,5 6 and the phosphorylation of MLC in the spastic anterior spinal artery is increased.7 In addition, the phosphatase activity toward myosin in the basilar artery is decreased in vasospasm.8 Because the use of a Ca2+ indicator in experiments of smooth muscle contraction revealed that the force/Ca2+ ratio in smooth muscle contraction is variable, the Ca2+/CaM-dependent MLC kinase pathway cannot solely account for the mechanisms of agonist-induced or GTP-
-S–induced 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-protein–mediated
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+
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Materials and Methods |
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Cerebral vasospasm was produced in dogs by an injection of 5 mL fresh autogenous arterial blood into the cisterna magna, followed by another injection 2 days later. Vasocontractions were induced by a topical application of 80 mmol/L KCl and 0.5 µmol/L serotonin for 15 minutes (KCl-15 and serotonin-15 subgroups, respectively) or 90 minutes (KCl-90 and serotonin-90 subgroups, respectively) to the canine basilar artery exposed via a transclival route. Vasospasm and vasocontraction of the basilar artery were confirmed by angiography or by use of a surgical microscope. The animals were euthanized after a perfusion with 500 mL physiological salt solution, and the basilar artery was removed together with the brain. In the vasospasm group, the blood clot around the basilar artery and its branches was carefully removed without any mechanical stimulation given to the arteries. After it was washed briefly with physiological salt solution, the basilar artery was quickly frozen in liquid nitrogen until used.
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 anti–glutathione-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 anti–glutathione-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.
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Results |
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Caliber of the Basilar Artery
No significant angiographic narrowing of the basilar artery was shown before the animals in the control group were killed. The percentage of caliber reduction, as shown in Figure 1
, indicates the occurrence of vasospasm
and the increase in its intensity with the lapse of time after SAH. The
mean percent calibers in the KCl and serotonin groups
decreased over a period of 
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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The phosphorylation of MLC in the basilar artery examined by glycerol-urea gel electrophoresis and subsequent immunoblot analysis demonstrated an increase in vasospasm with the passage of time after SAH. At the same time, Rho-kinase was activated, and the phosphorylation of MBS was elevated in the basilar artery in vasospasm. Recently, Kimura et al21 have shown that activated Rho-kinase phosphorylates MBS of myosin phosphatase, thereby leading to a decrease in myosin phosphatase activity in vitro. The decrease in activity of myosin phosphatase in the spastic basilar artery reported previously8 is consistent with the present increase in MBS phosphorylation levels. It has been shown that MBS is phosphorylated and that myosin phosphatase activity is inactivated during the action of thromboxane A2 in platelets, and both reactions are blocked by prior treatment of platelets with Clostridium botulinum C3 toxin, which interferes with Rho functions.28 Similar observations have been made in the endothelial cells responding to thrombin.29 In addition, the activated Rho-kinase stoichiometrically phosphorylates MLC at the Ser19 residue, which is the site phosphorylated by Ca2+/CaM-dependent MLC kinase.19 Thus, the activated Rho-kinase in vasospasm appears to induce MLC phosphorylation by the direct phosphorylation and/or inactivation of myosin phosphatase. It may be noted that the direct phosphorylation of MLC by Rho-kinase in smooth muscle remains to be elucidated, because GTP-
-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--S–induced 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 A–induced 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-27632–specific 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-27632–sensitive 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 Rho–Rho-kinase pathway is activated during the
development of cerebral vasospasm. How is the Rho–Rho-kinase pathway
activated? Several recent studies imply that some trimeric
G-protein–coupled receptors, including lysophosphatidic acid,
thrombin, and serotonin receptors, are linked to the
Rho–Rho-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
G13, 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 Rho–Rho-kinase pathway
remains to be clarified, it is tempting to speculate that certain
ligands for the G-protein–coupled 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-protein–coupled
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.
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Acknowledgments |
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This study was supported in part by grant-in-aid for Scientific Research No. 09877280 from the Ministry of Education, Science, Sports, and Culture, Japan. We thank Dr M. Amano, Dr Y. Fukata, and Dr Y. Kawano, Division of Signal Transduction, Nara Institute of Science and Technology, for their helpful discussion and critical comments on the manuscript. Y-27632 was kindly provided by Yoshitomi Pharmacentical Industrics, Ltd, Osaka, Japan.
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Footnotes |
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Reprint requests to Dr Eiichi Tani, Department of Neurosurgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. E mail fuji-ns@hyo-med.ac.jp
Received September 20, 1999; accepted June 9, 2000.
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 G. Y. Rochefort and E. D. Michelakis COUNTERPOINT: RELEASE OF AN ENDOTHELIUM-DERIVED VASOCONSTRICTOR AND RHOA/RHO KINASE-MEDIATED CALCIUM SENSITIZATION OF SMOOTH MUSCLE CELL CONTRACTION ARE NOT THE MAIN EFFECTORS FOR FULL AND SUSTAINED HPV J Appl Physiol, May 1, 2007; 102(5): 2072 - 2075. [Full Text] [PDF]
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T. A. Parker, G. Roe, T. R. Grover, and S. H. Abman Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L976 - L982. [Abstract] [Full Text] [PDF]
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 J. K. Liao Rho-Kinase: A Potential Link Between Hypercholesterolemia and Abnormal Vascular Smooth Muscle Contraction Circ. Res., August 4, 2006; 99(3): 238 - 239. [Full Text] [PDF]
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 S. Chrissobolis and C. G. Sobey Recent Evidence for an Involvement of Rho-Kinase in Cerebral Vascular Disease Stroke, August 1, 2006; 37(8): 2174 - 2180. [Abstract] [Full Text] [PDF]
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 K. Noma, N. Oyama, and J. K. Liao Physiological role of ROCKs in the cardiovascular system Am J Physiol Cell Physiol, March 1, 2006; 290(3): C661 - C668. [Abstract] [Full Text] [PDF]
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G. Loirand, P. Guerin, and P. Pacaud Rho Kinases in Cardiovascular Physiology and Pathophysiology Circ. Res., February 17, 2006; 98(3): 322 - 334. [Abstract] [Full Text] [PDF]
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Y. Chiba, M. Murata, H. Ushikubo, Y. Yoshikawa, A. Saitoh, H. Sakai, J. Kamei, and M. Misawa Effect of Cigarette Smoke Exposure In Vivo on Bronchial Smooth Muscle Contractility In Vitro in Rats Am. J. Respir. Cell Mol. Biol., December 1, 2005; 33(6): 574 - 581. [Abstract] [Full Text] [PDF]
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Y. P. R. Jarajapu and H. J. Knot Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1917 - H1922. [Abstract] [Full Text] [PDF]
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Y. Rikitake, H.-H. Kim, Z. Huang, M. Seto, K. Yano, T. Asano, M. A. Moskowitz, and J. K. Liao Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection Stroke, October 1, 2005; 36(10): 2251 - 2257. [Abstract] [Full Text] [PDF]
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 H. Shimokawa and A. Takeshita Rho-Kinase Is an Important Therapeutic Target in Cardiovascular Medicine Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1767 - 1775. [Abstract] [Full Text] [PDF]
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 Y. Rikitake and J. K. Liao Rho-Kinase Mediates Hyperglycemia-Induced Plasminogen Activator Inhibitor-1 Expression in Vascular Endothelial Cells Circulation, June 21, 2005; 111(24): 3261 - 3268. [Abstract] [Full Text] [PDF]
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 K. Budzyn, P. D. Marley, and C. G. Sobey Opposing Roles of Endothelial and Smooth Muscle Phosphatidylinositol 3-Kinase in Vasoconstriction: Effects of Rho-Kinase and Hypertension J. Pharmacol. Exp. Ther., June 1, 2005; 313(3): 1248 - 1253. [Abstract] [Full Text] [PDF]
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Y. Watanabe, F. M. Faraci, and D. D. Heistad Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2653 - H2658. [Abstract] [Full Text] [PDF]
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 K. Budzyn, P. D. Marley, and C. G. Sobey Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R342 - R348. [Abstract] [Full Text] [PDF]
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D. A. Emmert, J. A. Fee, Z. M. Goeckeler, J. M. Grojean, T. Wakatsuki, E. L. Elson, B. P. Herring, P. J. Gallagher, and R. B. Wysolmerski Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts Am J Physiol Cell Physiol, January 1, 2004; 286(1): C8 - C21. [Abstract] [Full Text] [PDF]
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C. Lan, D. Das, A. Wloskowicz, and B. Vollrath Endothelin-1 modulates hemoglobin-mediated signaling in cerebrovascular smooth muscle via RhoA/Rho kinase and protein kinase C Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H165 - H173. [Abstract] [Full Text] [PDF]
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 A. P. SOMLYO and A. V. SOMLYO Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase Physiol Rev, October 1, 2003; 83(4): 1325 - 1358. [Abstract] [Full Text] [PDF]
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 A. Cavarape, J. Bauer, E. Bartoli, K. Endlich, and N. Parekh Effects of angiotensin II, arginine vasopressin and tromboxane A2 in renal vascular bed: role of rho-kinase Nephrol. Dial. Transplant., September 1, 2003; 18(9): 1764 - 1769. [Abstract] [Full Text] [PDF]
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 K. Noma, Y. Higashi, D. Jitsuiki, K. Hara, M. Kimura, K. Nakagawa, C. Goto, T. Oshima, M. Yoshizumi, and K. Chayama Smoking Activates Rho-Kinase in Smooth Muscle Cells of Forearm Vasculature in Humans Hypertension, May 1, 2003; 41(5): 1102 - 1105. [Abstract] [Full Text] [PDF]
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G. Wickman, C. Lan, and B. Vollrath Functional Roles of the Rho/Rho Kinase Pathway and Protein Kinase C in the Regulation of Cerebrovascular Constriction Mediated by Hemoglobin: Relevance to Subarachnoid Hemorrhage and Vasospasm Circ. Res., April 18, 2003; 92(7): 809 - 816. [Abstract] [Full Text] [PDF]
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H. Teoh, M. Zacour, A. D. Wener, L. Gunaratnam, and M. E. Ward Increased myofibrillar protein phosphatase-1 activity impairs rat aortic smooth muscle activation after hypoxia Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1182 - H1189. [Abstract] [Full Text] [PDF]
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Z. Guo, W. Su, Z. Ma, G. M. Smith, and M. C. Gong Ca2+-independent Phospholipase A2 Is Required for Agonist-induced Ca2+ Sensitization of Contraction in Vascular Smooth Muscle J. Biol. Chem., January 10, 2003; 278(3): 1856 - 1863. [Abstract] [Full Text] [PDF]
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E. Q. Scherer, M. Herzog, and P. Wangemann Endothelin-1-Induced Vasospasms of Spiral Modiolar Artery Are Mediated by Rho-Kinase-Induced Ca2+ Sensitization of Contractile Apparatus and Reversed by Calcitonin Gene-Related Peptide Stroke, December 1, 2002; 33(12): 2965 - 2971. [Abstract] [Full Text] [PDF]
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M. Ishiguro, C. B. Puryear, E. Bisson, C. M. Saundry, D. J. Nathan, S. R. Russell, B. I. Tranmer, and G. C. Wellman Enhanced myogenic tone in cerebral arteries from a rabbit model of subarachnoid hemorrhage Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2217 - H2225. [Abstract] [Full Text] [PDF]
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F. Nakao, S. Kobayashi, K. Mogami, Y. Mizukami, S. Shirao, S. Miwa, N. Todoroki-Ikeda, M. Ito, and M. Matsuzaki Involvement of Src Family Protein Tyrosine Kinases in Ca2+ Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway Circ. Res., November 15, 2002; 91(10): 953 - 960. [Abstract] [Full Text] [PDF]
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L. Miao, Y. Dai, and J. Zhang Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H983 - H989. [Abstract] [Full Text] [PDF]
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A. V. Somlyo New Roads Leading to Ca2+ Sensitization Circ. Res., July 26, 2002; 91(2): 83 - 84. [Full Text] [PDF]
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S. Shirao, S. Kashiwagi, M. Sato, S. Miwa, F. Nakao, T. Kurokawa, N. Todoroki-Ikeda, K. Mogami, Y. Mizukami, S. Kuriyama, et al. Sphingosylphosphorylcholine Is a Novel Messenger for Rho-Kinase-Mediated Ca2+ Sensitization in the Bovine Cerebral Artery: Unimportant Role for Protein Kinase C Circ. Res., July 26, 2002; 91(2): 112 - 119. [Abstract] [Full Text] [PDF]
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K. G. Lamping Enhanced Contractile Mechanisms in Vasospasm: Is Endothelial Dysfunction the Whole Story? Circulation, April 2, 2002; 105(13): 1520 - 1522. [Full Text] [PDF]
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C. Kataoka, K. Egashira, S. Inoue, M. Takemoto, W. Ni, M. Koyanagi, S. Kitamoto, M. Usui, K. Kaibuchi, H. Shimokawa, et al. Important Role of Rho-kinase in the Pathogenesis of Cardiovascular Inflammation and Remodeling Induced by Long-Term Blockade of Nitric Oxide Synthesis in Rats Hypertension, February 1, 2002; 39(2): 245 - 250. [Abstract] [Full Text] [PDF]
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M. Tosaka, F. Okajima, Y. Hashiba, N. Saito, T. Nagano, T. Watanabe, T. Kimura, and T. Sasaki Sphingosine 1-Phosphate Contracts Canine Basilar Arteries In Vitro and In Vivo: Possible Role in Pathogenesis of Cerebral Vasospasm Stroke, December 1, 2001; 32(12): 2913 - 2919. [Abstract] [Full Text] [PDF]
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Z. Wang, N. Jin, S. Ganguli, D. R. Swartz, L. Li, and R. A. Rhoades Rho-Kinase Activation Is Involved in Hypoxia-Induced Pulmonary Vasoconstriction Am. J. Respir. Cell Mol. Biol., November 1, 2001; 25(5): 628 - 635. [Abstract] [Full Text] [PDF]
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 T. P Woodsome, M. Eto, A. Everett, D. L Brautigan, and T. Kitazawa Expression of CPI-17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle J. Physiol., September 1, 2001; 535(2): 553 - 564. [Abstract] [Full Text] [PDF]
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R. J. Solaro Myosin Light Chain Phosphatase : A Cinderella of Cellular Signaling Circ. Res., August 4, 2000; 87(3): 173 - 175. [Full Text] [PDF]
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K. E. Kamm and J. T. Stull Dedicated Myosin Light Chain Kinases with Diverse Cellular Functions J. Biol. Chem., February 9, 2001; 276(7): 4527 - 4530. [Full Text] [PDF]
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S. Chrissobolis and C. G. Sobey Evidence That Rho-Kinase Activity Contributes to Cerebral Vascular Tone In Vivo and Is Enhanced During Chronic Hypertension : Comparison With Protein Kinase C Circ. Res., April 27, 2001; 88(8): 774 - 779. [Abstract] [Full Text] [PDF]
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