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1 From the Departments of Ophthalmology and Visual Sciences and 2 Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan; the 3 Department of Ophthalmology, Tenri Hospital, Tenri, Japan; the 4 Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan; the 5 Department of Molecular Pathophysiology, Graduate School of Pharmaceutical Sciences, Osaka, Japan; and the 6 Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. A specific ROCK inhibitor Y-27632 was used. The IOP, the outflow facility, and the pupil diameter were determined before and after the topical, intracameral, or intravitreal administration of Y-27632 in rabbits. Western blot analysis was used to identify specific ROCK isoform in human trabecular meshwork (TM) cells and bovine ciliary muscle (CM) tissues. The cell morphology and distribution of actin filaments and vinculin in TM cells were studied by cell biology techniques. Carbachol (Cch)-induced contraction of isolated bovine CM strips after administration of Y-27632 was measured in a perfusion chamber.
RESULTS. In rabbit eyes, administration of Y-27632 resulted in a significant decrease in IOP in a dose-dependent manner. An increase of the outflow facility and pupil size dilation was also observed in Y-27632–treated eyes. Western blot analysis revealed the presence of p160ROCK in human TM cells and bovine CM tissues. In cultured human TM cells, exposure to Y-27632 caused retraction and rounding of cell bodies as well as disruption of actin bundles and impairment of focal adhesion formation. Y-27632 in addition inhibited Cch-induced contraction of isolated bovine CM strips.
CONCLUSIONS. Administration of Y-27632 caused a reduction in IOP and an increase in the outflow facility. The in vitro experiments suggest that the IOP-lowering effects of Y-27632 may be related to the altered cellular behavior of TM cells and relaxation of CM contraction. These studies suggest that ROCK inhibitors may have great potential to be developed for treatment of glaucoma and other ocular diseases.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/Rho kinase/ROCK
II.14
15
16
ROCKs play a key role in focal adhesions and
stress fiber formation in cultured fibroblasts and epithelial
cells,12
14
16
and in Ca2+
sensitization of smooth muscle cells.17
18
Y-27632 is a
recently identified specific inhibitor of the ROCK–ROK family of
protein kinases.19
This compound inhibits agonist-induced
smooth muscle contraction both in vitro and in vivo, as well as the
stress fibers and focal adhesion formation induced by p160ROCK in
cultured cells.19 Intraocular pressure (IOP) in the eye is regulated so it remains within the normal range. In abnormal situations such as in glaucoma,20 21 22 the IOP is often elevated, causing damage to the optic nerve head that leads to blindness. Because glaucoma is the second leading cause of blindness,23 extensive efforts have been made to develop antiglaucoma drugs and laser and other surgical procedures to lower IOP.23 Most of these modalities are intended either to modulate the aqueous outflow facility at sites of the trabecular meshwork (TM) and the ciliary muscle (CM),21 24 25 26 or to inhibit aqueous humor production by the ciliary body. Despite the widely recognized functional importance of TM and CM tissues in regulating the aqueous outflow, cellular mechanisms underlying these functions are not well understood. Previous studies have suggested that alteration in activities or behavior of TM cells may change the outflow facility.21 27 28 Also, modulation of the contractile or relaxant elements in CM may play a direct role in the regulation of aqueous humor outflow.25 Both TM and CM cells morphologically and electrophysiologically express properties that are typical of smooth muscle cells.29 Thus, alteration in the contractility of the TM and CM, and modulation of the cellular behavior of TM cells are the likely bases for the development of novel IOP-lowering antiglaucoma drugs. Y-27632 has been shown to induce inhibition of smooth muscle contraction and alter various cellular behavior19 30 and therefore appears to be a key candidate.
In the present study, we examined whether inhibition of Rho–ROCK signaling by a specific ROCK inhibitor Y-27632 causes an increase in the outflow facility. The data suggest that Y-27632 induced a significant decrease in IOP, possibly through an effect on TM cellular behavior.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals, Drug Preparation, and Drug Administration
Y-27632 (molecular weight 338.3) was supplied by Welfide
(formerly Yoshitomi Pharmaceutical, Saitama, Japan). The structure of
Y-27632 is shown in Figure 1
. For topical administration, Y-27632 (10 or 100 mM) or vehicle
phosphate buffered saline (PBS) was administered to the central cornea
as four 3-µl drops at intervals of 30 seconds, with blinking
prevented between and after the last drops. The Y-27632 dosage for
topical administration was chosen to provide a 10- or 100-µM Y-27632
concentration in the approximately 120-µl rabbit anterior
chamber,31
assuming 1% intracameral penetration and no
drug loss from the anterior chamber.32
33
After topical
anesthesia of rabbit eyes, 1.2 µl of 1, 10, or 100 mM of Y-27632 was
administered intracamerally (10, 100, and 1000 µM final
concentrations, respectively) and 14 µl of 10 or 100 mM Y-27632 was
administered intravitreally (100 and 1000 µM, respectively, in the
approximately 1.4-ml vitreous space to the rabbit eye).31
The fellow eyes that were treated with injection of appropriate volumes
of vehicle phosphate buffered saline (PBS) served as a control.
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IOP Measurement
A calibrated pneumotonometer (Alcon, Forth Worth, TX) was used
to monitor the IOP. It was measured before the administration of
Y-27632 and at 0.5, 1, 3, 6, 9, and 24 hours after administration.
PD Measurement
The pupil diameter (PD) was measured with a millimeter ruler
(Digimatic Caliper; Mitutoyo, Tokyo, Japan) under standard laboratory
light at the same time points at which the IOP measurements were made.
Total Outflow Facility and Uveoscleral Outflow
Total outflow facility was determined by two-level
constant-pressure perfusion (25 and 35 mm Hg) 3 hours after topical
administration of 12 µl of 100 mM Y-27632 and vehicle, according to
the method of Barany.34
Briefly, the anterior chambers of
rabbits anesthetized with 40% urethane were perfused with mock aqueous
humor (BSS plus; Santen Pharmaceutical, Osaka, Japan) by a constant
pressure of either 25 or 35 mm Hg, which was alternately applied at
10-minute intervals. During each 10-minute period, fluid flow was
measured for 8 minutes beginning 2 minutes after the pressure change
was induced.
Uveoscleral outflow was determined with a perfusion technique using
fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight
71,200; Sigma, St. Louis, MO)35
36
3 hours after the
topical administration of 12 µl of 100 mM Y-27632 and vehicle. The
rabbits were anesthetized with 40% urethane, and two 23-gauge needles
connected to a pair of syringes were inserted into the anterior chamber
in each eye of each rabbit. The pair of syringes was controlled by an
infusion–withdrawal pump (model 944; Harvard Apparatus, South Natick,
MA), and the infusion syringe was filled with
10-4 M FITC-dextran. One
milliliter of the FITC-dextran solution was washed through the anterior
chamber using the syringes at a rate of 0.5 ml/min. The IOP level was
then set to 20 mm Hg. The FITC-dextran solution was perfused
continuously through the anterior chamber at a rate of 10 µl/min for
30 minutes. The anterior chamber was washed with 2 ml of PBS at a rate
of 0.5 ml/min. Each eye was then enucleated and dissected into the
following sample groupings: anterior uvea, anterior sclera, posterior
sclera plus posterior uvea, and the posterior segment fluid plus
vitreous. All samples were homogenized and centrifuged, and the volume
of each was measured. The supernatant was measured to determine the
FITC-dextran concentration using a fluorophotometer. The uveoscleral
outflow (Fu, in microliters per
minute) was calculated as follows:
 | (1) |
Slit Lamp Biomicroscopy
The integrity of the corneal epithelium, the presence or absence
of anterior chamber flare or cells, and lens clarity were examined 1,
3, 6, 12, and 24 hours after the administration of Y-27632.
Culture of Human TM Cells
Human eyes from donors were obtained from the Illinois Eye Bank
(Chicago, IL). Trabecular tissues excised from eyes were cultured on
flasks (Falcon Primaria; Becton Dickinson, Lincoln Park, NJ), as
previously described.37
38
The culture medium included
Dulbecco’s minimum essential Eagle’s medium (DMEM), 10% fetal bovine
serum (FBS), and antibiotics. Cells were maintained in a 95% air–5%
CO2 atmosphere at 37°C and passaged using the
trypsin-EDTA method. Only well-characterized normal human TM cells from
passages three through eight were used for subsequent studies.
Chemicals
Carbachol (Cch), FITC-phalloidin, and mouse monoclonal antibody
to vinculin were obtained from Sigma. Rabbit anti-p160ROCK (ROCK I) was
prepared as described.39
Appropriate secondary antibodies
were obtained from Chemicon (Temecula, CA) and Amersham (Little
Chalfont, UK).
Preparation of Lysates from Whole-Cell and Bovine Tissue and
Immunoblot Analysis
To examine the expression levels of p160ROCK, detergent lysates
of TM cells and bovine CM tissue were prepared in Laemmli’s sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer. Confluent cultures of human TM cells were kept in FBS-free DMEM
overnight to rule out ROCK upregulation. They were placed on ice,
washed with ice-cold PBS three times, and then scraped and lysed.
Bovine CM tissues were excised from bovine eyes with fine forceps and
Vannas scissors under a dissecting microscope. The tissues were lysed,
and the total protein content was quantified by a protein assay (DC;
Bio-Rad, South Richmond, CA). Equivalent amounts of protein-containing
lysates were boiled in sample buffer and subjected to SDS-PAGE using a
5% to 20% gradient gel (Daiichi, Tokyo, Japan). After
electrophoresis, the proteins were electrophoretically transferred onto
polyvinylidene difluoride membranes (Millipore, Bedford, MA). The
membrane was blocked with 2% bovine serum albumin (BSA) in PBS
containing 0.005% Tween 20 (PBST) at 4°C for 16 hours and was
incubated in a 1:1000 dilution of anti-p160ROCK rabbit antibody
2049039
for 2 hours at room temperature. The membrane was
washed with PBST and further incubated with biotin-conjugated
anti-rabbit antibody (1:1000; Amersham) and ABC solution (ABC Elite
kit; Vector, Burlingame, CA). After extensive washing, the blotted
protein bands were visualized with immunostain (HRP-1000; Konica,
Tokyo, Japan).
Effects of Y-27632 on the Shape of Human TM Cells
In experiments designed to examine changes in cell shape, sparse
cultures (Fig. 4)
, and semiconfluent cultures (Figs. 5
6)
were
incubated with various concentrations of Y-27632 (10–100 µM), with
or without serum. The cultures were observed by phase-contrast
microscopy and photographed immediately after drug application, and
0.5, 1, 2, and 18 hours later. The drug solution was removed afterward
and replaced with plain DMEM containing 10% FBS. In all cases,
recovery of normal morphology was documented 24 hours later.
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Measurement of Contractility of CM
Enucleated bovine eyes were obtained from a local slaughterhouse
and placed on ice. Small CM strips were carefully dissected according
to procedures described by Lepple–Wienhues et al.40
Briefly, after excision of the iris, meridional CM strips were excised
perpendicular to the circular ciliary body. The isolated strips were
approximately 5 mm long and 1 to 2 mm wide. The CM contractility was
measured isometrically with a force-length transducer device. The
bovine CM strips were set between a hook and an isometric force
transducer connecting an amplifier and a multipen recorder and
vertically mounted in a 20-ml Magnus tube (Iwashiyakishimoto,
Kyoto, Japan) filled with Krebs-Henseleit solution, which was aerated
continuously. The composition of the solution was NaCl, 118.1 mM; KCl,
4.7 mM; CaCl2 2.5 mM;
KH2PO4, 1.2 mM;
MgSO4, 1.34 mM; NaHCO3,
25.0 mM; and glucose, 11.1 mM; and the temperature was kept at 37°C.
At the beginning of each experiment, tissues were subjected to an
imposed resting tension and allowed to equilibrate for 120 minutes. To
confirm the stability of the preparations, application of Cch and
washing were repeated twice. Only strips that showed a stable tone were
used for experiments. After reproducible responses to Cch were
obtained, bovine CM strips were contracted with Cch that was applied to
the bath (10-6 M). When
Cch-induced contraction became stable (approximately 5 minutes after
the application), Y-27632 was added cumulatively to the bath. After the
final dose of Y-27632, the strip was perfused with plain Krebs solution
(to reestablish baseline tension). Mean data of isometric force
measurements were expressed as relative values in comparison with the
maximum Cch concentration
(10-6 M) response.
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-measures analysis of variance
(ANOVA) and Bonferroni as a post hoc test of the time course of IOP.
Mann–Whitney test, or Student’s t-test with the Bonferroni
correction was used for aqueous humor dynamics. P <
0.05 was considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurements of Outflow Facility
The outflow facility was measured 3 hours after topical
administration of Y-27632 when maximal IOP reduction was
observed. Results summarized in Table 1
showed that the average outflow facility was approximately two times
higher (+100%) in the eyes treated with 100 mM Y-27632 (0.24 ±
0.02 µl/min per millimeter mercury, P < 0.001) than
that in the contralateral PBS-treated control eyes (0.12 ± 0.01
µl/min per millimeter of mercury). The uveoscleral outflow was also
increased by 30% in the treated eyes (0.60 ± 0.05 µl/min)
compared with the control eyes (0.46 ± 0.04 µl/min), although
the difference was not statistically significant.
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Effects of Y-27632 on Cytoskeleton of Cultured Human TM Cells
To examine whether the actin structure was also affected, 10, 100,
or 1000 µM Y-27632 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 (Fig. 6)
. In control cells, actin filaments were assembled into large radial
and circumferential bundles in association with focal adhesions (Fig. 6A)
. After treatment with 100 µM Y-27632 for 30 minutes, the cells
lost most of their actin bundles (Fig. 6B)
. Instead of discrete long
filament beams, the phalloidin staining took on a punctate appearance,
as residual actin filaments became associated with the cell periphery
rather than focal adhesions. The cell borders and processes were
outlined distinctly. This change appeared to coincide with the rounding
of the cell shape.
Vinculin in control cells was predominantly associated with focal adhesions (Fig. 6A) . After the Y-27632 treatment, deterioration of focal adhesions was evident (Fig. 6B) . These cytoskeletal changes were completely reversible within 2 hours and completely recovered after 15 hours. These data suggest that the inhibition of the Rho–ROCK pathway may initiate the rearrangement of cytoskeleton in TM cells.
Measurement of Contractility of Isolated CM
After adjustment of baseline tension, Cch at a concentration of
10-6 M was used to induce
contraction in isolated CM strips, as described
previously.42
43
Figure 7
shows a typical recording of the relaxation effects induced by
cumulatively added Y-27632. Superfusion with Cch resulted in an
immediate, steep-force development, reaching maximum after 3 minutes.
Y-27632 led to relaxation of the Cch precontracted CM strips, in a
dose-dependent manner. The maximal effect was found in experiments
using 10-3 M Y-27632,
which almost completely abolished (by 97%) the response to Cch. Figure 8 summarizes the data obtained with increasing concentrations of Y-27632.
At 10-5 to
10-3 M of concentrations,
the Y-27632–induced relaxation of the Cch-precontracted CM strips was
significant. The average relaxation for Y-27632-treated CM strips was
12.2% ± 4.9%, 25.6% ± 7.7%, 43.2% ± 5.0%, 60.9% ± 3.1%, and
96.7% ± 0.6%, for 10-7,
10-6,
10-5,
10-4, and
10-3 M Y-27632, with
P = 0.6332, 0.1297, 0.0032, 0.0006, and < 0.0001,
respectively, compared with the time-matched control samples.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoblot analysis showed that a specific isoform p160ROCK is present in cultured human TM cells and bovine CM tissues. The p160ROCK isoform, a Rho effector and a target molecule of Y-27632, has been reported to play an important role in smooth muscle contractility and actin cytoskeletal integrity. It appears that TM and CM are both target tissues of Y-27632.
There are two routes of aqueous humor outflow: conventional (trabecular) and unconventional (uveoscleral) pathways.21 In human and primate eyes, conventional outflow is regarded as the main route37 38 44 and is believed to be regulated by the cellular behavior and contractility of both TM and CM cells.45 Our outflow facility data suggest that Y-27632 may affect the conventional outflow. The presence of p160ROCK in cultured TM cells and CM tissues further support this conjecture.
In rabbit eyes, the anterior chamber has no true trabecula, and the vascular anatomy of the outflow pathways and orbit differs from that of the primate.31 The rabbit has a venous plexus in intimate association with the chamber angle tissues and a large orbital venous sinus; there is no Schlemm’s canal or collector channel arrangement as in primates. The significant IOP-lowering effect of Y-27632 found in rabbit eyes thus may be related not only to alterations in the trabecular facility, but also to changes in the permeability of the chamber angle venous plexus and/or the iris vasculature. The two-level constant pressure perfusion system used in our experiments for the outflow facility measurements monitors changes in flow from an elevated external reservoir into the anterior chamber that represent total facility—i.e., the sum of conventional and unconventional outflow facility. Y-27632 induced a twofold increase in the total outflow facility. Our measurements of unconventional outflow revealed a modest increase in the uveoscleral outflow in rabbit eyes by Y-27632, but its effects were not statistically significant. These results suggest that IOP-lowering effects of Y-27632 are largely mediated by the altered conventional facility.
Exposure of the cultured TM cells to 10 µM of Y-27632-induced retraction and rounding of cell bodies, as well as disruption of actin microfilament bundles and impairment of focal adhesion formation. These morphologic and cytoskeletal events were similar to those reported previously in other cell types such as N1E-115 and Swiss 3T3 cells.41 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 10 to 1000 µM Y-27632.
It has been shown previously that the Rho–ROCK signaling pathways acts, at least in part, by controlling the phosphorylation of myosin light chain (MLC) and ultimately the organization of the actin cytoskeleton through the actomyosin system.46 This mechanism of action contrasts with that of cytochalasin and latrunculins, which are effective IOP-reducing agents as well, but with disassembly of the cytoskeletal actin as the primary event.28 Other compounds such as protein kinase inhibitor staurosporine, and serine-threonine kinase inhibitor H-7, which can decrease outflow resistance,47 48 also cause cytoskeletal perturbation as their common feature. Similar to the ROCK inhibitor, however, the actin effect only occurs secondarily through interference of the actomyosin interaction.49 Between the different compounds even in similar categories, there are still differences in effects and mechanisms.
Y-27632 also reduces contractility in cultured neuronal cells, nonneuronal cells, and fibroblasts.43 The contraction of the actomyosin system in smooth muscle and nonmuscle cells is thought to be regulated by two mechanisms: the increase in free Ca2+ ion in the cell mediated by MLC kinase and a Ca2+ sensitization mechanism. Several lines of evidence now indicate a role for the ROCK-ROK family in the latter mechanism.16 17 19 In our experiments, Y-27632 led to relaxation of bovine CM strips in a dose-dependent manner. Many investigators have reported that relaxation of CM would not increase trabecular outflow. Thus, at present, we cannot conclude that Y-27632-induced changes in CM contribute to hypotensive effects of this drug.
In summary, the present study shows that Y-27632, a selective ROCK inhibitor, reduces IOP and increases the outflow facility. Such effects may be related to altered cellular behavior of TM cells and relaxation of CM tissues. Selective inhibition of the Rho–ROCK signaling pathway may be developed into a novel neuroprotective strategy for the treatment of glaucoma and retinal ischemic diseases.
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Footnotes |
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Submitted for publication March 20, 2000; revised August 3 and September 21, 2000; accepted September 29, 2000.
Commercial relationships policy: N.
Corresponding author: Hidenobu Tanihara, Department of Ophthalmology, Tenri Hospital, Mishima-cho 200, Tenri, 632-0015, Japan. tanihara{at}pearl.ocn.ne.jp
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) 10 mM; and (
) 100
mM Y-27632. For (b) and (c) (•) vehicle alone; (
) 100 mM Y-27632. The results are presented as mean ±
SEM [n = 6 for (a), (b) and (c)]. The significance of
the data was evaluated by Student’s unpaired t-test;
*P < 0.05 and **P < 0.01,
P < 0.005, 
P < 0.001, compared
with controls with vehicle alone.
