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Effects of Protein Kinase Inhibitor, HA1077, on Intraocular Pressure and Outflow Facility in Rabbit Eyes
Megumi Honjo, MD;
Masaru Inatani, MD;
Noriaki Kido, MD;
Tatsuya Sawamura, MD;
Beatrice Y.-J. T. Yue, MD;
Yoshihito Honda, MD;
Hidenobu Tanihara, MD
Arch Ophthalmol. 2001;119:1171-1178.
ABSTRACT
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Objective To elucidate the roles of protein kinase in regulating the intraocular
pressure (IOP) and outflow facility in rabbit eyes.
Materials and Methods A protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-homopiperazine
(HA1077), was used. The IOP and the outflow facility were measured before
and after topical, intracameral, or intravitreal administration of HA1077
in rabbits. Western blot analysis was performed to detect the 20-kd light
chain of myosin in human trabecular meshwork (TM) cells and bovine ciliary
muscle (CM) tissues. The cell morphologic condition and distribution of actin
filaments and vinculin in TM cells were studied using cell biology techniques.
Carbachol-induced contraction of isolated bovine CM strips following administration
of HA1077 was examined in a perfusion chamber.
Results In rabbit eyes, the administration of HA1077 resulted in a significant
decrease in IOP in a dose-dependent manner. An increased outflow facility
was also observed. Western blot analysis revealed the presence of 20-kd light
chain of myosin in human TM cells and bovine CM tissues. In cultured human
TM cells, exposure to HA1077 disrupted actin bundles and impaired focal adhesion
formation. In addition HA1077 showed relaxation of bovine CM strips.
Conclusions Use of HA1077 caused a reduction in IOP and an increase in the outflow
facility. The results of in vitro experiments suggest that the IOP-lowering
effects of HA1077 may be related to the altered cellular behavior of TM cells
and relaxation of CM contraction. The results of these studies suggested that
protein kinase inhibitors have the potential to be developed into a treatment
modality for glaucoma.
INTRODUCTION
IN GLAUCOMATOUS eyes, elevation of intraocular pressure (IOP) is believed
to be one of the major factors that causes axonal damage in the optic nerve
head and the subsequent retinal ganglion cell death, leading to blindness.1-2 The IOP is regulated essentially through
2 routes of the aqueous humor outflow—conventional (trabecular) and
unconventional (uveoscleral) pathways.3 Conventional
outflow, the major pathway, is influenced by the cellular behaviors and cell-cell
junctions of trabecular meshwork (TM) cells.4
In the TM system, series of investigations have indicated that the alterations
in the contractility and cellular behaviors of TM cells can affect the IOP
and the aqueous outflow.1, 3, 5-9
An antivasospastic compound, 1-(5-isoquinolinesulfonyl)-homopiperazine
(HA1077), has been previously shown to act as a vasodilator in vivo and is
currently used for the treatment of cerebral vasospasm, inhibiting agonist-induced
smooth muscle contraction.10 This compound
has also been shown to be able to induce inhibition of smooth muscle contraction
and alter various cellular behaviors.11-12
Rho GTPase, a member of the Rho subgroups of the Ras superfamily, is
involved in diverse physiological functions associated with cytoskeletal rearrangements,13-14 such as cell morphology,15 cell motility,16 cytokinesis,17 and smooth muscle contraction.18-19
Recently, several putative target molecules of the Rho have been identified
as Rho effectors, including p160ROCK,20-21
ROK /Rho kinase/ROCK II,22-24
and protein kinase N (PKN).20 ROCK has been
shown to phosphorylate the largest subunit of myosin phosphatase in the carboxyl
terminal region, resulting in inhibition of the phosphatase activity.25-26 This inhibition is suggested to be
responsible for the Rho-mediated Ca2+-sensitization process.27-28 The ROCK-mediated inhibition of myosin
phosphatase also accounts for an increase in the 20-kd light chain of myosin
(MLC) phosphorylation, and the resultant contractility of actomyosin is proposed
to induce stress fibers and focal adhesions.25-26,29
In addition to the inhibition of myosin phosphatase, ROCK has also been reported
to directly phosphorylate MLC in vitro.30 1-(5-Isoquinolinesulfonyl)-homopiperazine
has been reported to inhibit the activity of both ROCK and PKN.11, 31-32
Compounds that interfere with actomyosin action such as the protein kinase
inhibitor including ROCK inhibitor may be potential targets for the development
of novel IOP-lowering antiglaucoma drugs. In this study, we examined the effects
of a protein kinase inhibitor, HA1077, on the IOP and aqueous outflow facility
in rabbits and its influence on the TM cellular behavior in culture.
MATERIALS AND METHODS
ANIMALS AND ANESTHESIA
Adult Japanese white rabbits, weighing 2 to 2.5 kg, were used in this
study. All studies were conducted in accord with the Association of Vision
Research in Ophthalmology Statement for the Use of Animals in Ophthalmic and
Vision Research. For IOP measurements, the rabbit eyes were anesthetized by
topical instillation of 2% lidocaine hydrochloride. For measurements of the
outflow facility or the uveoscleral outflow, the rabbits were anesthetized
with 40% urethane (1.0-1.5 mL/kg).
CHEMICALS, DRUG PREPARATION, AND DRUG ADMINISTRATION
The HA1077 was supplied by Asahi Chemical Industry Co Ltd, Tokyo, Japan.
Carbachol (CCh), flourescein isothiocyanate conjugated–phalloidin, mouse
monoclonal antibody to vinculin, and MLC were obtained from Sigma Chemical
Co, St Louis, Mo. Appropriate secondary antibodies were obtained from Chemicon
International, Temecula, Calif. For topical administration, HA1077 (1 or 10
mmol/L) was administered as four 3-µL drops, to give a 1- or 10-µmol/L
concentration in the approximately 120-µL rabbit anterior chamber33 assuming 1% intracameral penetration and no drug
loss from the anterior chamber.34-35
After topical anesthesia of rabbit eyes, 1.2 µL of 0.1-, 1-, or 10-mmol/L
HA1077 was administered intracamerally (1-, 10-, or 100-µmol/L final
concentration, respectively) and 14 µL of 1- or 10-mmol/L HA1077 was
administered intravitreally (10- and 100-µmol/L final concentration,
respectively) in the approximately 1.4-mL vitreous space in the rabbit eye.33 The fellow eyes that were treated with vehicle phosphate-buffered
solution served as controls.
IOP MEASUREMENT AND SLITLAMP BIOMICROSCOPY
A calibrated pneumotonometer (Alcon, Fort Worth, Tex) was used to measure
IOP. Intraocular pressure was monitored before the administration of HA1077,
and at 0.5-, 1-, 3-, 6-, 9-, 12-, and 24-hour time points after the administration.
The integrity of the corneal epithelium, the presence or absence of anterior
chamber flare or cells, and lens clarity were also noted.
TOTAL OUTFLOW FACILITY AND UVEOSCLERAL OUTFLOW
Total outflow facility and uveoscleral outflow were measured as follows.
Total outflow facility was determined by 2-level constant pressure perfusion
(25 and 35 mm Hg) 3 hours after topical administration of 100-mmol/L HA1077
or vehicle, according to the method of Bárány36
and Taniguchi et al.37 Uveoscleral outflow
was determined with a perfusion technique using fluorescein isothiacyanate
conjugated–dextran (molecular weight = 71 200, Sigma Chemical Co)37-38 beginning at 3 hours after the topical
administration of 100-mmol/L HA1077 or vehicle. The amount of tracer in the
tissues was measured using a fluorophotometer.
CULTURE OF HUMAN TM CELLS
Human eyes from donors were obtained from the Illinois Eye Bank, Chicago.
Trabecular tissues excised from eyes were cultured in flasks (Falcon Primaria;
Becton Dickson, Lincoln Park, NJ) as previously described.39-40
The culture medium included Dulbecco Eagle minimum essential medium, 10% fetal
bovine serum (FBS), and antibiotic agents. Cells were maintained in a 95%
room air and 5% carbon dioxide atmosphere at 37°C and passaged using the
trypsin-EDTA method. Only well-characterized normal human TM cells from passages
3 through 8 were used for subsequent studies.
PREPARATION OF LYSATES FROM WHOLE CELL AND BOVINE TISSUE AND IMMUNOBLOTTING
To examine the expression of MLC, detergent lysates of TM cells and
bovine CM tissue were prepared in Laemmli sodium dodecyl sulfate–polyacrylamide
gel electrophoresis sample buffer, and subjected to sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. After electrophoresis, the proteins were electrophoretically
transferred onto polyvinylidene difluoride membranes (Millipore Co, Bedford,
Mass) and incubated serially with primary and secondary antibodies. The blotted
protein bands were visualized with an immunostain (Konica immunostain HRP-1000;
Konica, Tokyo, Japan).
EFFECTS OF HA1077 ON CELL SHAPE OF HUMAN TM CELLS
In experiments designed to examine changes in cell shape, postconfluent
and semiconfluent TM cultures were incubated with various concentrations of
HA1077 (1-1000 µmol/L) with or without serum. The cultures were observed
by phase-contrast microscopy and photographed immediately after drug application,
and 10, 30, and 60 minutes later. The drug solution was removed afterward
and replaced with plain Dulbecco Eagle minimum essential medium containing
10% FBS. In all cases, recovery of normal morphology was documented 2 and
15 hours later.
ACTIN AND VINCULIN STAINING
Human TM cells were plated on coverglasses at a density of 3 x
104 cells per each 6-cm dish. After culturing for 2 days, when
cells reached semiconfluence, HA1077 was added and incubated. For controls,
phosphate-buffered saline solution was added as a vehicle. After the drug
exposure, the cells on coverglasses were fixed with 3% paraformaldehyde–phosphate-buffered
saline solution and 0.5% Triton X-100 (Sigma Chemical Co) for 20 minutes.
Filamentous actin (F-actin) was labeled with fluorescein isothiocyanate conjugated–phalloidin
(0.05 mg/mL) for 1 hour. For vinculin staining, the coverglasses were incubated
successively with antivinculin antibody (1:400) for 1 hour and with secondary
antibody for 30 minutes. Fluorescence was visualized under an epifluorescence
microscope (Zeiss Axioplan, Oberkochen, Germany) and with a confocal laser
scanning microscope (Bio-Rad, Hercules, Calif). To determine whether the effects
of HA1077 were reversible, the cells were incubated for another 30 minutes
in HA1077-free medium after the various HA1077 treatments, fixed, and stained.
MEASUREMENT OF CONTRACTILITY OF CM
Enucleated bovine eyes were obtained from a local slaughterhouse and
placed on ice. Small bovine CM strips were carefully dissected according to
procedures described by Lepple-Wienhues et al.41
Briefly, after excision of the iris, meridional CM strips were excised. The
CM contractility was measured isometrically with a force-length transducer
device using an isometric force transducer connecting an amplifier, a multipen
recorder, and vertically mounted in a 20-mL Magnus tube filled with continuously
aerated Krebes-Hensleit solution. Only CM strips that showed a stable tone
were used for experiments. The HA1077 was added cumulatively to the bath.
Relaxation responses were expressed as a percentage of the maximum effect
(100%) elicited by CCh in each strip.
STATISTICAL ANALYSIS
Data were analyzed by repeated measured analysis of variance and Bonferroni
adjustment as a post hoc test of time course of IOP. Mann-Whitney test was
used for aqueous humor dynamics. P<.05 was considered
to be statistically significant.
RESULTS
IOP MEASUREMENT AND SLITLAMP BIOMICROSCOPY
Compared with contralateral vehicle-treated controls, the IOP in rabbit
eyes was significantly (P<.01) lowered at 0.5
hour following topical administration of a 10-µmol/L concentration of
HA1077 eyedrops. The IOP reduction was maximally observed at 3 hours with
a 10-µmol/L concentration of HA1077 (Figure 1A). After intravitreal administration, significant IOP reductions
were noted between 0.5 and 12 hours, and the maximal reductions were seen
between 3 and 6 hours with the 100-µmol/L concentration (P<.001) (Figure 1B). When
administered intracamerally, significant IOP reductions occurred between 0.5
and 12 hours and 100-µmol/L HA1077 produced the maximal reductions (P<.001) (Figure 1C).
No anterior chamber, lens, or fundus abnormalities in rabbit eyes were detected
by slitlamp examination following either the topical, intracameral, or intravitreal
administration of HA1077. These experiments, thus, demonstrated the potent
IOP-lowering effects of HA1077 in rabbit eyes.
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Figure 1. Effects of HA1077 on the intraocular
pressure (IOP). The HA1077 was administered into rabbit eyes topically, intravitreally,
and intracamerally. The contralateral eyes were treated with the same volume
of vehicle phosphate-buffered saline solution topical administration (A),
intravitreal administration (B), and intracameral administration (C). A, Solid
circle indicates vehicle alone; open circle, 1-µmol/L concentration
of HA1077; and triangle, 10-µmol/L concentration of HA1077. B-C, Solid
circle indicates vehicle alone; open circle, 1-µmol/L concentration
of HA1077; triangle, 10-µmol/L concentration of HA1077; and square,
100-µmol/L concentration of HA1077. The results are presented as mean
± SEM (n = 6 for A-C). The statistical significance of the data was
evaluated by unpaired ttest. Single asterisk indicates P<.05; double asterisks, P<.01;
dagger, P<.005; and double dagger, P<.001 compared with controls with vehicle alone.
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MEASUREMENTS OF THE OUTFLOW FACILITY
The outflow facility was measured 3 hours after topical administration
of HA1077 when maximal IOP reduction was observed. Results summarized in Table 1 showed that the average outflow
facility was approximately 2-fold higher in the eyes treated with the 10-µmol/L
concentration of HA1077 (0.27 ± 0.03 µL/min per millimeters of
mecury, P<.005) than that in the contralateral
phosphate-buffered saline solution–treated control eyes (0.12 ±
0.01 µL/min per millimeters of mecury). The uveoscleral outflow was
also increased by 17% in the treated eyes (0.55 ± 0.04 µL/min)
compared with the control eyes (0.47 ± 0.03 µL/min), although
the difference was not statistically significant.
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Effects of HA1077 on Outflow Facility in the Rabbit Eye*
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WESTERN BLOT ANALYSIS FOR THE IDENTIFICATION OF MLC IN TM CELLS AND
CM
A series of in vitro experiments were carried out to elucidate the mechanisms
of the IOP-lowering and outflow facility-enhancing effects of HA1077 observed
in animals. As shown in Figure 2,
Western blot analysis using anti-MLC antibody detected a protein band of approximately
20 kd in both intact bovine CM tissue (lane 2) and cultured human TM cells
(lane 4). This molecular size corresponded to that reported for MLC.
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Figure 2. Western blot analysis of light
chain of myosin (MLC) in bovine ciliary muscle and cultured human trabecular
meshwork (TM) cells. Homogenate of bovine ciliary muscle tissue and whole
cell lysates from cultured human TM cells were run in sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. Lanes 1 and 3 indicate control mouse IgG; lanes 2 and
4, MLC.
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EFFECTS OF HA1077 ON MORPHOLOGY OF CULTURED HUMAN TM CELLS
Next, the morphology of TM cells was examined. By phase-contrast microscopy,
treatment with 100-µmol/L HA1077 in the presence of serum for 30 minutes
induced retraction and rounding of TM cells (Figure 3). When semiconfluent cultures were treated with HA1077,
TM cells also retracted and became thinner (Figure 4B; upper 4 rows). To determine whether such changes were
related to the Rho/ROCK pathway by serum stimulation, the cells were also
incubated in serum-free medium. Retraction and thinning were seen 30 to 60
minutes later (Figure 4B, bottom
row). These results showed that the TM morphology might be influenced by inhibition
of the Rho/ROCK signaling system.
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Figure 3. Effect of HA1077 on trabecular
meshwork (TM) cell characteristics. Effects of HA1077 on cell morphology.
Human TM cells from sparse culture prior (A) to and after (B) a 30-minute
incubation with 100-µmol/L HA1077. Note the marked cell thinning (original
magnification x100).
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Figure 4. Effects of HA1077 on morphology
of cultured human trabecular meshwork (TM) cells. Phase-contrast microscopic
observation of semiconfluent culture of human TM cells. Treatments with HA1077
in concentrations of 1 µmol/L (upper row), 10 µmol/L (upper middle
row), 100 µmol/L (middle row), and 1000 µmol/L (lower middle row)
for 10, 30, and 60 minutes in the presence of serum resulted in retraction
and thinning of the cells (arrows). Serum starvation also resulted in a similar
cell shape change with retraction and thinning of the cells (lower row; arrow).
The drug solution or Dulbecco Eagle minimum essential medium without serum
was removed afterward and replaced with Dulbecco Eagle minimum essential medium
containing 10% fetal bovine serum. Recovery of normal morphologic status was
observed 2 and 15 hours later (original magnification x60).
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EFFECTS OF HA1077 ON CYTOSKELETON OF CULTURED HUMAN TM CELLS
To examine whether the actin structure was affected, 1-, 10-, 100-,
or 1000-µmol/L HA1077 was added to the culture medium. It was found
that the distribution of F-actin was altered dramatically in a time- and concentration-dependent
manner (Figure 5). In control cells,
actin filaments were assembled into large radial and circumferential bundles
in association with focal adhesions (Figure
5A). As Figure 5B shows,
HA1077 produced distinctive effects on the microfilament organization in TM
cells. Treatment with 100-µmol/L HA1077 for 30 minutes caused loss of
most of their actin bundles in TM cells (Figure 5B). After treatment with 10-µmol/L HA1077 for 60 minutes,
the stress fibers in the center of TM cells were labeled with phalloidin;
however, the peripheral bundles were lost. Vinculin in control cells was predominantly
associated with focal adhesions (Figure 5A). After the HA1077 treatment, deterioration of focal adhesions
in the cell periphery was evident (Figure
5B). These cytoskeletal changes were reversible within 2 hours,
and completely recovered after 15 hours.
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Figure 5. Distribution of filamentous actin
(F-actin) and vinculin in human trabecular meshwork (TM) cells treated with
HA1077. A, Distribution of F-actin (in green) and vinculin (in red) in normal
human TM cells. Small white arrows show F-actin bundles, and white arrowheads
show focal adhesions associated with vinculin. a, Confocal images. b, Cells
were stained with antibody to vinculin. c, Cells were stained with flourescein
isothiocyanate conjugated–phalloidin to visualize F-actin. B, Distribution
of F-actin and vinculin in human TM cells treated with HA1077 in concentrations
of 1, 10, 100, and 1000 µmol/L for 10, 30, and 60 minutes. The drug
solutions were removed afterward and replaced with Dulbecco Eagle minimum
essential medium containing 10% fetal bovine serum. Recovery of normal morphologic
status was observed 2 and 15 hours later. White arrows point to F-actin bundles,
which disappeared with HA1077 treatment and recovered after drug removal.
White arrowheads show vinculin-containing focal adhesions that were decreased
with HA1077 treatment and recovered by replacement with Dulbecco Eagle minimum
essential medium. Bar indicates 10 µm.
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MEASUREMENT OF CONTRACTILITY OF ISOLATED CM
After adjustment of baseline tension, CCh at a concentration of
10-6mol/L was used to induce contraction in isolated bovine CM strips
as described previously.42-43 Figure 6 shows a typical recording of the
relaxation effects induced by cumulatively added HA1077. Superfusion by the
CCh resulted in an immediate steep force development that reached maximum
after 3 minutes. The HA1077 led to relaxation of the CCh precontracted bovine
CM strips in a dose-dependent manner. The maximum effect was found in experiments
using 10-3-mol/L HA1077, which almost completely abolished
(by 99%) the response to CCh. Recovery of contractility in the presence of
CCh after the removal of HA1077 was also observed. Figure 7 shows the data obtained with increasing concentrations
of HA1077. At 10-5- to 10-3-mol/L concentrations,
the HA1077-induced relaxation of the CCh precontracted bovine CM strips was
significant. The average ± SD relaxation for HA1077-treated bovine
CM strips was 6.5% ± 1.0%, 10.8% ± 1.1%, 28.5% ± 5.8%,
42.5% ± 2.6%, 99.3% ± 0.5 %, for 10-7-, 10-6-, 10-5-, 10-4-, and 10-3-mol/L HA1077, with P values being
.33, .78, .02, .001, and <.001, respectively, compared with the time-matched
controls.
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Figure 6. Representative recordings of isometric
force developed in isolated strips of bovine ciliary muscle strips. After
a carbachol (10-6-mol/L)-induced contraction, HA1077 led
to relaxation in the isolated bovine ciliary muscle. When the carbachol response
became stable, HA1077 was cumulatively added to the bovine ciliary muscle
strip.
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Figure 7. Dose-dependent relaxation of isolated
ciliary muscle strips by HA1077. Carbachol was applied to isolated bovine
ciliary muscle strips (n = 4). The percentage of maximum carbachol response
is shown with increasing concentrations of HA1077 plus 10-6-mol/L
carbachol. Data are given as mean ± SEM; the 4 ciliary muscle strips
are from 4 bovine eyes. Solid circle indicates vehicle alone; open circle,
HA1077.
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COMMENT
The present study demonstrated that a protein kinase inhibitor, HA1077,
when administered topically, intracamerally, or intravitreally, induces a
significant decrease in IOP in rabbit eyes. To elucidate the mechanisms of
the IOP-lowering effects of this inhibitor, we have conducted a series of
experiments.
First, physiologic experiments showed that HA1077 elicits changes in
the total outflow facility, but not in the unconventional one. Conventional
outflow is the main route in human and primate eyes,39, 44
and is believed to be regulated by the cellular behavior of both CM and TM
cells.4 In rabbit eyes, the anterior chamber
lacks a true "trabeculum," and the outflow pathologic anatomy differs from
that of the primate.33 There is no Schlemm
canal or collector channel arrangement as in the primates, and the rabbit
has a venous plexus in intimate association with the chamber angle tissues
and a large orbital venous sinus. The significant IOP-lowering effect of HA1077
found in rabbit eyes, thus, may be related to not only alterations in the
trabecular facility, but also to changes in the permeability of the chamber
angle venous plexus and/or the iris vasculature. Our data suggested that the
IOP-lowering effect of this compound is related to increased conventional
outflow.
Second, our cytochemical studies demonstrated that HA1077 disrupted
F-actin bundles and impaired focal adhesion formation in the cultured TM cells.
Similar findings have been reported previously on the serine-threonine–kinase
inhibitor, H-7, which also disrupts cell junctions and results in a decrease
in IOP.6, 45 The outflow resistance
is decreased by H-7 and it also causes cytoskeletal perturbation. The kinetics
of the alterations in the cultured TM cells paralleled that of the observed
IOP and outflow facility changes in animal eyes after administration of 1
to 100-µmol/L HA1077. It has been shown that ROCK, an effector of Rho,
acts downstream of Rho resulting in inhibition of myosin phosphatase and consequent
enhancement of MLC phosphorylation.11, 25-26,29
Light chain of myosin phosphorylation is known to be a mechanism that controls
the actomyosin contractility in many cell lines 27, 46-47
and is reported to be essential and sufficient for the formation of stress
fibers and focal adhesions in fibroblastic cells.48
It has been previously shown that HA1077 inhibits Rho-mediated enhancement
of Ca2+-induced MLC phosphorylation.11
It was also demonstrated previously that HA1077 inhibits not only ROCK but
also PKN, another Rho-associated protein kinase. However, it was demonstrated
that in vitro PKN neither phosphorylated myosin phosphatase nor inhibited
its activity.49 These observations are consistent
with the notion that the effects of HA1077 on TM cell integrity may be related
to cytoskeletal changes induced by the alteration in balance of MLC phosphorylation
mediated by ROCK. Our immunoblot results showed that MLC is present in cultured
human TM cells; this also supports our hypothesis.
Furthermore, in our experiments using bovine CM strips, HA1077 led to
relaxation of the smooth muscle in a dose-dependent manner. Many investigators
reported that relaxation of CM would not increase trabecular outflow. Although
similar findings have been reported in previous studies, we are unable to
conclude that HA1077-induced changes in CM contribute to the hypotensive effects
of this drug. Further studies will be required to assess the role of CM relaxation
in the IOP-lowering effects of HA1077.
In summary, this study shows that HA1077, a protein kinase inhibitor,
reduces IOP and increases outflow facility. Such effects may be related to
altered cellular behavior of TM cells. Inhibition of the Rho signaling pathway
may be developed into a new strategy for the treatment of glaucoma.
AUTHOR INFORMATION
Accepted for publication February 23, 2001.
This investigation was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, and Culture, Tokyo,
Japan, from the Ministry of Health and Welfare, Tokyo, and grants EY 05628
and EY 01792 from the National Eye Institute, National Institutes of Health,
Bethesda, Md.
Dr Honjo is a recipient of a Fellowship of the Japan Society for the
Promotion of Sciences for Young Scientists.
We thank Asahi Chemical Industry Co Ltd, Tokyo, for supplying HA1077.
Corresponding author: Hidenobu Tanihara, Department of Ophthalmology,
Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan
(e-mail: tanihara{at}pearl.ocn.ne.jp).
From the Department of Ophthalmology and Visual Sciences, Kyoto University
Graduate School of Medicine, Kyoto, Japan (Drs Honjo, Inatani, Kido, and Honda);
Department of Bioscience, National Cardiovascular Center Research Institute
and the Department of Molecular Pathophysiology, Graduate School of Pharmaceutical
Sciences, Osaka University, Osaka, Japan (Dr Sawamura); Department of Ophthalmology
and Visual Sciences, University of Illinois at Chicago, College of Medicine
(Dr Yue); and the Department of Ophthalmology, Kumamoto University School
of Medicine, Kumamoto, Japan (Dr Tanihara).
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