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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 t test. Single asterisk indicates P<.05; double asterisks, P<.01; dagger, P<.005; and double dagger, P<.001 compared with controls with vehicle alone.

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 t test. Single asterisk indicates P<.05; double asterisks, P<.01; dagger, P<.005; and double dagger, P<.001 compared with controls with vehicle alone.

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.

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.

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 ×100).

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 ×100).

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 ×60).

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 ×60).

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.

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.

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.

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.

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.

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.

Effects of HA1077 on Outflow Facility in the Rabbit Eye*
Effects of HA1077 on Outflow Facility in the Rabbit Eye*
1.
Kaufman  PLGabelt  BTCynader  M Introductory comments on neuroprotection. Surv Ophthalmol. 1999;43suppl 1S89- S90Article
2.
Hart  WM Intraocular pressure. Hart  WMedAdler's Physiology of the Eye: Clinical Application 9th St Louis, Mo Mosby–Yearbook Inc1992;248- 467
3.
Kaufman  PL Pressure-dependent outflow. Hart  WMedAdler's Physiology of the Eye: Clinical Application 9th St Louis, Mo Mosby–Yearbook Inc1992;307- 335
4.
Epstein  DLRohen  JW Morphology of the trabecular meshwork and inner-wall endothelium after cationized ferritin perfusion in the monkey eye. Invest Ophthalmol Vis Sci. 1991;32160- 171
5.
Maepea  OBill  A The pressures in the episcleral veins, Schlemm's canal and the trabecular meshwork in monkeys: effects of changes in intraocular pressure. Exp Eye Res. 1989;49645- 663Article
6.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;116633- 643Article
7.
Tian  BGeiger  BEpstein  DLKaufman  PL Cytoskeletal involvement in the regulation of aqueous humor flow. Invest Ophthalmol Vis Sci. 2000;41619- 623
8.
Wiederholt  MBielka  SSchweig  FLutjen-Drecoll  ELepple-Wienhues  A Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye. Exp Eye Res. 1995;61223- 234Article
9.
Honjo  MTanihara  HInatani  M  et al.  Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001;42137- 144
10.
Asano  MSuzuki  TTsuchiya  M  et al.  Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol. 1989;981091- 1100Article
11.
Nagumo  HSasaki  YOno  YOkamoto  HSeto  MTakuwa  Y Rho kinase inhibitor HA1077 prevents Rho-mediated myosin phosphatase inhibition in smoooth muscle cells. Am J Physiol Cell Physiol. 2000;278C57- C65
12.
Szaski  KKurashima  KKapus  A  et al.  Rho A and Rho kinase regulate the epithelial Na+/H+exchange NHE3: role of myosin light chain phosphorylation. J Biol Chem. 2000;27528599- 28606Article
13.
Narumiya  SIshizaki  TWatanabe  N Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 1997;41068- 72Article
14.
Narumiya  S The small GTPase Rho: cellular functions and signal transduction. J Biochem (Tokyo). 1996;120215- 228Article
15.
Paterson  HFSelf  AJGarrett  MDJust  IAktories  KHall  A Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990;1111001- 1007Article
16.
Takaishi  KSasaki  TKato  M  et al.  Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene. 1994;9273- 279
17.
Kishi  KSasaki  TKuroda  SItoh  TTakai  Y Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol. 1993;1201187- 1195Article
18.
Hirata  KKikuchi  ASasaki  T  et al.  Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem. 1992;2678719- 8722
19.
Gong  MCIizuka  KNixon  G  et al.  Role of guanine nucleotide-binding proteins—ras-family or trimeric proteins or both—in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci U S A. 1996;931340- 1345Article
20.
Ishizaki  TNaito  MFujisawa  K  et al.  p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 1997;404118- 124Article
21.
Nakagawa  OFujisawa  KIshizaki  TSaito  YNakao  KNarumiya  S ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett. 1996;392189- 193Article
22.
Leung  TChen  XQManser  ELim  L The p160 Rho A-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 1996;165313- 5327
23.
Matsui  TAmano  MYamamoto  T  et al.  Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP-binding protein Rho. EMBO J. 1996;152208- 2216
24.
Amano  MChihara  KKimura  K  et al.  Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science. 1997;2751308- 1311Article
25.
Kimura  KIto  MAmano  M  et al.  Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996;273245- 248Article
26.
Kawano  YFukata  YOshiro  N  et al.  Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol. 1999;1471023- 1038Article
27.
Somlyo  APSomlyo  AV Signal transduction and regulation in smooth muscle. Nature. 1994;372231- 236[published correction appears in Nature. 1994;372:812]Article
28.
Hartshorne  DJIto  MErdodi  F Myosin light chain phosphatase: subunit composition, interactions, and regulation. J Muscle Res Cell Motil. 1998;19325- 341Article
29.
Chrzanowska-Wodnicka  MBurridge  K Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;1331403- 1415Article
30.
Amano  MIto  MKimura  K  et al.  Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem. 1996;27120246- 20249Article
31.
Sward  KDreja  KSusnjar  MHellstrand  PHartshorne  DJWalsh  MP Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol (Lond). 2000;52233- 49Article
32.
Amano  MChihara  KNakamura  NKaneko  TMatsuura  YKaibuchi  K The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity. J Biol Chem. 1999;27432418- 32424Article
33.
Poyer  JFGabelt  BKaufman  PL The effect of topical PGF2-α on uveoscleral outflow and outflow facility in the rabbit eye. Exp Eye Res. 1992;54277- 283Article
34.
Janes  RGStiles  JF The penetration of C14-labeled atropine into the eye: a comparison of methods of application. Arch Ophthalmol. 1959;6269- 74Article
35.
Asseff  CFWeisman  RLPodos  SMBecker  B Ocular penetration of pilocarpine in primates. Am J Ophthalmol. 1973;75212- 215
36.
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in vervet monkeys by constant pressure perfusion. Invest Ophthalmol. 1964;2135- 143
37.
Taniguchi  THaque  MSRSugiyama  KHori  NKitazawa  Y Ocular hypotensive mechanism of topical isopropyl unoprostone, a novel prostaglandin metabolite-regulated drug, in rabbits. J Ocul Pharmacol Ther. 1996;12489- 498Article
38.
Takashima  YTaniguchi  TYoshida  MHaque  MSYoshimura  NHonda  Y Ocular hypotensive mechanism of intravitreally injected brain natriuretic peptide in rabbit. Invest Ophthalmol Vis Sci. 1996;372671- 2677
39.
Yue  BYHigginbotham  EJChang  IL Ascorbic acid modulates the production of fibronectin and laminin by cells from an eye tissue-trabecular meshwork. Exp Cell Res. 1990;18765- 68Article
40.
Sawaguchi  SYue  BYChang  ILWong  FHigginbotham  EJ Ascorbic acid modu aerated lates collagen type I gene expression by cells from an eye tissue–trabecular meshwork. Cell Mol Biol. 1992;38587- 604
41.
Lepple-Wienhues  AStahl  FWiederholt  M Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle. Exp Eye Res. 1991;5333- 38Article
42.
Wiederholt  MSturm  ALepple-Wienhues  A Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci. 1994;352515- 2520
43.
Wiederholt  MGroth  JStrauss  O Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility. Invest Ophthalmol Vis Sci. 1998;391012- 1020
44.
Yue  BYLin  CCFei  PFTso  MO Effects of chondroitin sulfate on metabolism of trabecular meshwork. Exp Eye Res. 1984;3835- 44Article
45.
Epstein  DLRowlette  LLRoberts  BC Acto-myosin drug effects and aqueous outflow function. Invest Ophthalmol Vis Sci. 1999;4074- 81
46.
Kamm  KEStull  JT The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25593- 620Article
47.
Moussavi  RSKelley  CAAdelstein  RS Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains. Mol Cell Biochem. 1993;127-128219- 227ReviewArticle
48.
Totsukawa  GYamakita  YYamashiro  SHartshorne  DJSasaki  YMatsumura  F Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol. 2000;150797- 806Article
49.
Ishizaki  TMaekawa  M  et al.  The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 1996;151885- 1893
Laboratory Sciences
August 2001

Effects of Protein Kinase Inhibitor, HA1077, on Intraocular Pressure and Outflow Facility in Rabbit Eyes

Author Affiliations

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).

Arch Ophthalmol. 2001;119(8):1171-1178. doi:10.1001/archopht.119.8.1171
Abstract

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.

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.1,3,59

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 ROKα/Rho kinase/ROCK II,2224 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 × 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.

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.

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.

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.

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.

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.42,43Figure 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.

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.

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Article 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@pearl.ocn.ne.jp).

References
1.
Kaufman  PLGabelt  BTCynader  M Introductory comments on neuroprotection. Surv Ophthalmol. 1999;43suppl 1S89- S90Article
2.
Hart  WM Intraocular pressure. Hart  WMedAdler's Physiology of the Eye: Clinical Application 9th St Louis, Mo Mosby–Yearbook Inc1992;248- 467
3.
Kaufman  PL Pressure-dependent outflow. Hart  WMedAdler's Physiology of the Eye: Clinical Application 9th St Louis, Mo Mosby–Yearbook Inc1992;307- 335
4.
Epstein  DLRohen  JW Morphology of the trabecular meshwork and inner-wall endothelium after cationized ferritin perfusion in the monkey eye. Invest Ophthalmol Vis Sci. 1991;32160- 171
5.
Maepea  OBill  A The pressures in the episcleral veins, Schlemm's canal and the trabecular meshwork in monkeys: effects of changes in intraocular pressure. Exp Eye Res. 1989;49645- 663Article
6.
Tian  BKaufman  PLVolberg  TGabelt  BTGeiger  B H-7 disrupts the actin cytoskeleton and increases outflow facility. Arch Ophthalmol. 1998;116633- 643Article
7.
Tian  BGeiger  BEpstein  DLKaufman  PL Cytoskeletal involvement in the regulation of aqueous humor flow. Invest Ophthalmol Vis Sci. 2000;41619- 623
8.
Wiederholt  MBielka  SSchweig  FLutjen-Drecoll  ELepple-Wienhues  A Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye. Exp Eye Res. 1995;61223- 234Article
9.
Honjo  MTanihara  HInatani  M  et al.  Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility. Invest Ophthalmol Vis Sci. 2001;42137- 144
10.
Asano  MSuzuki  TTsuchiya  M  et al.  Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol. 1989;981091- 1100Article
11.
Nagumo  HSasaki  YOno  YOkamoto  HSeto  MTakuwa  Y Rho kinase inhibitor HA1077 prevents Rho-mediated myosin phosphatase inhibition in smoooth muscle cells. Am J Physiol Cell Physiol. 2000;278C57- C65
12.
Szaski  KKurashima  KKapus  A  et al.  Rho A and Rho kinase regulate the epithelial Na+/H+exchange NHE3: role of myosin light chain phosphorylation. J Biol Chem. 2000;27528599- 28606Article
13.
Narumiya  SIshizaki  TWatanabe  N Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 1997;41068- 72Article
14.
Narumiya  S The small GTPase Rho: cellular functions and signal transduction. J Biochem (Tokyo). 1996;120215- 228Article
15.
Paterson  HFSelf  AJGarrett  MDJust  IAktories  KHall  A Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990;1111001- 1007Article
16.
Takaishi  KSasaki  TKato  M  et al.  Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene. 1994;9273- 279
17.
Kishi  KSasaki  TKuroda  SItoh  TTakai  Y Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol. 1993;1201187- 1195Article
18.
Hirata  KKikuchi  ASasaki  T  et al.  Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem. 1992;2678719- 8722
19.
Gong  MCIizuka  KNixon  G  et al.  Role of guanine nucleotide-binding proteins—ras-family or trimeric proteins or both—in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci U S A. 1996;931340- 1345Article
20.
Ishizaki  TNaito  MFujisawa  K  et al.  p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett. 1997;404118- 124Article
21.
Nakagawa  OFujisawa  KIshizaki  TSaito  YNakao  KNarumiya  S ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett. 1996;392189- 193Article
22.
Leung  TChen  XQManser  ELim  L The p160 Rho A-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 1996;165313- 5327
23.
Matsui  TAmano  MYamamoto  T  et al.  Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP-binding protein Rho. EMBO J. 1996;152208- 2216
24.
Amano  MChihara  KKimura  K  et al.  Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science. 1997;2751308- 1311Article
25.
Kimura  KIto  MAmano  M  et al.  Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996;273245- 248Article
26.
Kawano  YFukata  YOshiro  N  et al.  Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol. 1999;1471023- 1038Article
27.
Somlyo  APSomlyo  AV Signal transduction and regulation in smooth muscle. Nature. 1994;372231- 236[published correction appears in Nature. 1994;372:812]Article
28.
Hartshorne  DJIto  MErdodi  F Myosin light chain phosphatase: subunit composition, interactions, and regulation. J Muscle Res Cell Motil. 1998;19325- 341Article
29.
Chrzanowska-Wodnicka  MBurridge  K Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol. 1996;1331403- 1415Article
30.
Amano  MIto  MKimura  K  et al.  Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem. 1996;27120246- 20249Article
31.
Sward  KDreja  KSusnjar  MHellstrand  PHartshorne  DJWalsh  MP Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol (Lond). 2000;52233- 49Article
32.
Amano  MChihara  KNakamura  NKaneko  TMatsuura  YKaibuchi  K The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity. J Biol Chem. 1999;27432418- 32424Article
33.
Poyer  JFGabelt  BKaufman  PL The effect of topical PGF2-α on uveoscleral outflow and outflow facility in the rabbit eye. Exp Eye Res. 1992;54277- 283Article
34.
Janes  RGStiles  JF The penetration of C14-labeled atropine into the eye: a comparison of methods of application. Arch Ophthalmol. 1959;6269- 74Article
35.
Asseff  CFWeisman  RLPodos  SMBecker  B Ocular penetration of pilocarpine in primates. Am J Ophthalmol. 1973;75212- 215
36.
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in vervet monkeys by constant pressure perfusion. Invest Ophthalmol. 1964;2135- 143
37.
Taniguchi  THaque  MSRSugiyama  KHori  NKitazawa  Y Ocular hypotensive mechanism of topical isopropyl unoprostone, a novel prostaglandin metabolite-regulated drug, in rabbits. J Ocul Pharmacol Ther. 1996;12489- 498Article
38.
Takashima  YTaniguchi  TYoshida  MHaque  MSYoshimura  NHonda  Y Ocular hypotensive mechanism of intravitreally injected brain natriuretic peptide in rabbit. Invest Ophthalmol Vis Sci. 1996;372671- 2677
39.
Yue  BYHigginbotham  EJChang  IL Ascorbic acid modulates the production of fibronectin and laminin by cells from an eye tissue-trabecular meshwork. Exp Cell Res. 1990;18765- 68Article
40.
Sawaguchi  SYue  BYChang  ILWong  FHigginbotham  EJ Ascorbic acid modu aerated lates collagen type I gene expression by cells from an eye tissue–trabecular meshwork. Cell Mol Biol. 1992;38587- 604
41.
Lepple-Wienhues  AStahl  FWiederholt  M Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle. Exp Eye Res. 1991;5333- 38Article
42.
Wiederholt  MSturm  ALepple-Wienhues  A Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci. 1994;352515- 2520
43.
Wiederholt  MGroth  JStrauss  O Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility. Invest Ophthalmol Vis Sci. 1998;391012- 1020
44.
Yue  BYLin  CCFei  PFTso  MO Effects of chondroitin sulfate on metabolism of trabecular meshwork. Exp Eye Res. 1984;3835- 44Article
45.
Epstein  DLRowlette  LLRoberts  BC Acto-myosin drug effects and aqueous outflow function. Invest Ophthalmol Vis Sci. 1999;4074- 81
46.
Kamm  KEStull  JT The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25593- 620Article
47.
Moussavi  RSKelley  CAAdelstein  RS Phosphorylation of vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains. Mol Cell Biochem. 1993;127-128219- 227ReviewArticle
48.
Totsukawa  GYamakita  YYamashiro  SHartshorne  DJSasaki  YMatsumura  F Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol. 2000;150797- 806Article
49.
Ishizaki  TMaekawa  M  et al.  The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 1996;151885- 1893
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