Blebbistatin, a Novel Inhibitor of Myosin II ATPase Activity, Increases Aqueous Humor Outflow Facility in Perfused Enucleated Porcine Eyes

Abstract

purpose. To investigate the specific role of myosin II, a critical biochemical determinant of cellular contraction, in modulation of aqueous humor outflow facility through the trabecular meshwork (TM) pathway.

methods. Expression of the nonmuscle myosin II heavy chains (IIA, IIB, and IIC) in human TM and ciliary body (CB) cells was determined by RT-PCR analyses. The effects of inhibition of myosin II on cell morphology, actomyosin organization, and cell adhesions were evaluated in porcine TM and CB cells treated with blebbistatin, a cell-permeable, specific inhibitor of myosin II adenosine triphosphatase (ATPase) activity. Changes in aqueous humor outflow facility were determined in enucleated porcine eyes by using a constant-pressure Grant perfusion model system. Ultrastructural integrity of the outflow pathway in drug-perfused eyes was analyzed by transmission electron microscopy.

results. Expression of nonmuscle myosin IIA and IIB was confirmed in both human TM and CB cells. Confluent cultures of primary porcine TM and CB cells treated with blebbistatin in the presence of serum revealed dose (10–200 μM)-dependent changes in cell morphology, decreases in actin stress fiber content and in focal adhesions and adherens junctions. These changes were found to be reversible within 24 hours of drug withdrawal from the cell culture media. Blebbistatin did not affect the status of myosin light chain phosphorylation in TM cells. Perfusion of enucleated porcine eyes for 5 hours with 100 and 200 μM blebbistatin produced a significant increase (P < 0.01, n = 7) in aqueous outflow facility (53% and 64%, respectively) from the baseline facility, compared with a 21% facility increase in sham control specimens. The integrity of the inner wall of aqueous plexi in drug-perfused porcine eyes was found to be intact, and TM cell morphology appeared to be similar to that noted in sham-treated eyes.

conclusions. These data demonstrate that selective inhibition of myosin II in the aqueous humor outflow pathway leads to increased aqueous outflow facility, suggesting a critical role for myosin II in the regulation of aqueous humor outflow facility. This study also suggests myosin II as a potential therapeutic target for lowering intraocular pressure in patients with glaucoma.

Increased intraocular pressure resulting from impaired aqueous humor drainage from the anterior chamber of the eye is a major risk factor for primary open-angle glaucoma. ^{1 2 3} The trabecular meshwork (TM) and Schlemm’s canal (SC) constitute a conventional pathway through which most of the aqueous humor is drained ^{3 4} and cells of the aqueous outflow pathway tissue, including the TM, SC, and ciliary body (CB) play important roles in homeostasis of aqueous humor outflow and maintenance of intraocular pressure. ^{3 5 6} However, the mechanistic basis of regulation of aqueous humor outflow in normal and glaucomatous eyes is not thoroughly understood. 

Cell morphology, cell–cell and cell–extracellular matrix (ECM) interactions and the contractile properties of cells of the aqueous outflow pathway have been suggested to modulate aqueous humor outflow through the TM. ^{6 7 8 9 10 11 12 13 14 15 16 17 18} Actomyosin interactions are thought to play a fundamental role in determining cell morphology, cell-ECM interactions, and contractile properties of various cell types. ^{19 20 21} Myosin II is a critical determinant of contractile characteristics and of cell motility and cell adhesion in several tissue types. ^{22 23 24} Myosin II activity is primarily regulated by the phosphorylation status of its regulatory subunit, also referred to as the myosin light chain (MLC). ^{20 22 24} Myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), regulate MLC phosphorylation in response to both calcium-dependent and -independent intracellular signaling pathways. ^{20 22 24} Direct and indirect pharmacological inhibitors of MLCK and MLCP have been reported to influence aqueous humor outflow facility in in vitro and in vivo models. ^{8 14 15 16 17 20 25 26 27 28 29 30 31 32} Perfusion of enucleated porcine eyes with inhibitors of protein kinase C (PKC), Rho kinase (ROCK), and MLCK has been documented to increase aqueous outflow facility by decreasing MLC phosphorylation. ^{8 17 20 28 31 32 33 34 35} In contrast, perfusion of certain physiological agents that increase MLC phosphorylation in TM cells has been reported to decrease aqueous humor outflow facility. ^{27 28} Collectively, these data suggest that the phosphorylation status of MLC in TM cells correlates inversely with changes in aqueous humor outflow facility and has a central role in the regulation of aqueous outflow through the TM. ²⁸ Thus, although many previous studies have implicated myosin in modulation of aqueous outflow through the trabecular pathway, ^{8 25 28 31 34 35 36} the specific role of myosin II in the regulation of aqueous humor outflow has not been directly addressed. 

Blebbistatin (a 1-phenyl-1-2-pyrrolidinone derivative; Fig. 1 ) was recently characterized as a specific pharmacologic inhibitor of skeletal muscle and nonmuscle myosin II adenosine triphosphatase (ATPase) activity. ^{37 38} It is a cell-permeable selective inhibitor with minimal effects on activity of smooth muscle myosin II or other types of myosin. ³⁹  

Furthermore, it has been demonstrated to be a more potent and selective inhibitor of myosin II than is 2,3-butanedione-2-monoxime (BDM), a commonly used inhibitor of myosin. ³⁹ Perfusion of enucleated porcine eyes with BDM has been documented to increase aqueous outflow facility ⁸ ; however, BDM has a broad substrate specificity, inhibiting various isoforms of myosin as well as MLCK. ^{40 41} In addition, BDM is a very poor inhibitor, with effective concentrations in the millimolar range. ⁸ Therefore, the specific effects of myosin II on outflow facility are yet to be determined. The purpose of this study was to determine the mechanistic bases underlying the role of myosin II in aqueous humor outflow facility. Our findings reveal a critical role of myosin II in modulating aqueous outflow facility, in that inhibition of nonmuscle myosin II in cells of the aqueous humor outflow pathway increases aqueous humor outflow facility, thereby suggesting a potential and a novel therapeutic avenue for lowering increased intraocular pressure in patients with glaucoma. 

Materials and Methods

Blebbistatin was procured from EMD Biosciences (La Jolla, CA); monoclonal antibodies against vinculin and β-catenin were from Sigma-Aldrich (St. Louis, MO) and Zymed (South San Francisco, CA), respectively; a polyclonal antibody against phospho-MLC from Cell Signaling Technology (Beverly, MA); and cell culture medium and fetal bovine serum (FBS) from Invitrogen-Gibco (Grand Island, NY). The human TM tissue cDNA library was generously provided by Pedro Gonzalez (Duke University School of Medicine). Enhanced chemiluminescence (ECL) detection reagents were from GE Healthcare (Piscataway, NJ). All other chemicals were of analytical grade. 

Cell Cultures

Porcine primary TM and CB cells were isolated from freshly obtained cadaveric eyes by collagenase IV digestion, as described previously. ¹⁷ The human eyes were obtained and managed according to the guidelines in the Declaration of Helsinki. Cells were cultured at 37°C in 5% CO₂, in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). All experiments were conducted by using confluent cell cultures between passages 3 and 5. 

Polymerase Chain Reaction Assays

To determine the expression pattern of nonmuscle myosin IIA, IIB, and IIC in TM and CB, reverse-transcribed total RNA generated from human TM cells (HTM; two sets, each derived from eyes of distinct donors, aged 26 and 54 years), CB cells (derived from a 69-year-old donor), and an HTM tissue cDNA library (donor age, 69 years) were amplified by PCR, by using sequence-specific forward and reverse oligonucleotide primers (Table 1) . A reverse-transcriptase–null sample (−RT) served as the negative control for RT-PCR. PCR products were resolved by agarose gel electrophoresis in conjunction with ethidium bromide for UV visualization. PCR-generated DNA products were also sequenced to confirm identity. 

Similarly, as described earlier, using human myosin IIA and IIB oligonucleotides described in Table 1 , we attempted to determine the expression profile of myosin IIA and IIB in porcine TM tissue by RT-PCR analysis. 

Actin and Cell Adhesions Staining

Porcine TM and CB cells were grown to confluence on gelatin–coated (2%) glass coverslips in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Cells were then treated with blebbistatin (10–200 μM dissolved in dimethyl sulfoxide [DMSO]) for 2 hours. The DMSO concentration was kept below 0.5% in the culture medium, for both drug treated and control cells. Changes in cell shape were recorded with a phase-contrast microscope (IM 35; Carl Zeiss Meditec, GmbH, Oberkochen, Germany). After treatment with blebbistatin, cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at room temperature, as described previously. ²⁷ Actin was stained with rhodamine-phalloidin, whereas focal adhesions and adherens junctions were stained with primary antibodies raised against vinculin and β-catenin, respectively, followed by the use of tetramethylrhodamine isothiocyanate–conjugated (TRITC) conjugated secondary antibodies. Micrographs were recorded by fluorescence microscope (Axioplan-II ; Carl Zeiss Meditec, GmbH). 

Cell Viability and Reversibility of Drug-Induced Effects

To evaluate the effects of blebbistatin on the viability of porcine TM cells, cells were grown to confluence on gelatin-coated glass coverslips. After treatment with either 100 or 200 μM of blebbistatin for 5 hours, cells were rinsed twice with PBS and then treated with fluorescein diacetate and propidium iodide for 3 minutes, as described by Erickson-Lamy et al. ⁹ Viable cells and dead or damaged cells, which stain green and red, respectively, were counted under a fluorescence microscope. 

To evaluate the reversibility of drug-induced effects, cells treated with blebbistatin (either a 100- or 200-μM concentration) for 5 hours were rinsed with cell culture medium a minimum of three times before the drug containing medium was replaced with regular cell culture medium. The reversibility of changes in cell shape was observed as a function of time after drug removal, using phase-contrast microscopy. After either a 24- or a 48-hour period after drug washout, cells were fixed and stained for actin filaments and focal adhesions, as described earlier. 

Myosin Light-Chain Phosphorylation

Myosin light-chain phosphorylation status in porcine TM cells was determined after the procedure described by Garcia et al. ⁴² Confluent cultures of porcine TM cells were treated with 25, 50, or 100 μM of blebbistatin for 2 hours, in the presence of serum. Control and drug-treated cells were then extracted with 10% cold trichloroacetic acid, and protein precipitates obtained after centrifugation at 13,000 rpm were dissolved in 8 M urea buffer containing 20 mM Tris, 23 mM glycine, 10 mM dithiothreitol (DTT), saturated sucrose, and 0.004% bromophenol, with a sonicator. Equal amounts of protein from the urea-solubilized samples were separated on slab gels containing 10% acrylamide, 0.5% bisacrylamide, 40% glycerol, 20 mM Tris, and 23 mM glycine. The running buffer for these gels comprised 20 mM Tris, 23 mM glycine, 2 mM DTT, and 2.3 mM thioglycolate in the upper chamber of the electrophoresis apparatus, whereas the lower chamber contained the same buffer without DTT and thioglycolate. Proteins from these glycerol gels were transferred onto nitrocellulose filters in 10 mM sodium phosphate buffer (pH 7.6), using a transfer apparatus (Bio-Rad, Hercules, CA). Nitrocellulose membranes were subjected to Western blot analysis with a phosphospecific myosin light chain (thr18/ser19) antibody. Blots were developed by using peroxidase-conjugated goat anti-rabbit IgG and an chemiluminescence detection system (ECL; GE Healthcare). Total protein concentration was determined with protein dye reagent, according to the manufacturer’s instructions (Bio-Rad). 

Aqueous Humor Outflow Facility

Freshly obtained enucleated porcine eyes were perfused with 25, 100, or 200 μM of blebbistatin using a Grant constant-pressure perfusion system. ¹⁷ Initial baseline outflow measurements were established at 15 mm Hg and 25°C with the perfusion medium containing Dulbecco’s PBS (DPBS; pH 7.4) and 5.5 mM d-glucose. After this, the anterior chambers of the test eyes were perfused with 25, 100, or 200 μM blebbistatin dissolved in DPBS containing DMSO (0.72%). Contralateral fellow sham eyes were perfused with DPBS containing DMSO alone for 5 hours. Outflow measurements were recorded at hourly intervals. Drug effects were expressed as the percentage of change in outflow facility (compared with the corresponding baseline percentages) over 5 hours, in drug- versus sham-treated paired control specimens. Data are expressed as the mean ± SE and were analyzed by a paired two-tailed Student’s t-test to determine significance. 

Histologic Analysis

At the end of a 5-hour perfusion period, two sets of sham control and drug-treated fellow eyes were fixed for histologic examination, by perfusing them with 2.5% glutaraldehyde and 2% formaldehyde at 15 mm Hg pressure at room temperature for 16 hours. Tissue quadrants obtained from drug-treated and control eyes were fixed in 1.0% osmium tetra oxide in 0.1 M sodium cacodylate buffer and then stained with 1% uranyl acetate. Finally, microtome-derived sections (70 nm) were stained sequentially with KMnO₄ and Sato’s stain and photographed by electron microscope (Jem-1200 EX; JEOL, Tokyo, Japan), as described previously. ¹⁷  

Results

Expression of Nonmuscle Myosin II in TM and CB Cells

To determine the expression profile of nonmuscle myosin II heavy-chain isoforms in human TM, CB cells, and TM tissue, we performed PCR analysis with reverse-transcribed total RNA, either extracted from human TM and CB cells or from a human trabecular meshwork tissue-derived cDNA library. PCR amplification yielded a DNA product of the expected size, confirming expression of myosin II A (MYH9) in TM and CB cells. cDNA derived from human TM tissue, in contrast, did not yield a detectable levels of myosin IIA specific product (Fig. 2A) . Myosin II B (MYH10) expression was evident in both TM tissue and TM cells and also in CB cells. PCR products were sequenced to confirm identity of nonmuscle myosin IIA and IIB. Based on relative abundance of PCR-amplified products, nonmuscle myosin IIA appeared to be abundantly expressed in both TM and CB cells. In contrast, we were unable to amplify myosin IIC (MYH14)-specific DNA product in TM, CB cells, or TM tissues (Fig. 2A) . Similar results were observed in a second batch of human TM cells derived from a different donor eye (data not shown). 

An extensive search of the GenBank and other sequence databases did not reveal a porcine-specific sequence for myosin IIA or IIB. However, to determine the expression profile of nonmuscle myosin IIA and IIB, we attempted amplifying myosin IIA and IIB from porcine TM tissues, using oligonucleotide primers directed against the corresponding human sequences. RT-PCR generated porcine TM tissue cDNA libraries (two independent samples) that yielded an easily detectable DNA product for myosin IIB. The DNA product was sequenced to confirm the identity, and BLAST sequence identity analysis revealed the highest sequence homology of the 200-bp DNA product with that of mouse myosin IIB (BLAST is provided in the public domain by NCBI, Bethesda, MD, and is available at www.ncbi.nlm.nih.gov/blast/). These data confirm the expression of nonmuscle IIB in porcine TM tissue (Fig. 2B) . In contrast, human myosin IIA oligonucleotide primers did not yield any DNA product, indicating possibly significant sequence differences between the human and porcine genes for myosin IIA. 

Effects of Blebbistatin on Actomyosin Organization and Cell Adhesions in Porcine TM and CB Cells

Treatment of confluent cultures of porcine TM and CB cells with blebbistatin (10–200 μM) for 2 hours induced changes in cell morphology. Drug-treated cells exhibited a dose-dependent progressive cell rounding and detachment, starting from 30 minutes after addition of the drug. Figure 3 , which depicts blebbistatin-induced changes in actin cytoskeletal organization and cell adhesions, also reveals changes in cell morphology. Blebbistatin treatment led to dose-and time-dependent decreases in actin stress fibers (phalloidin staining) in both TM (Fig. 3A)and CB cells (Fig. 3B) . Similarly, both focal adhesions (vinculin staining) and adherens junctions (β-catenin staining) were decreased in response to treatment with blebbistatin in both TM and CB cells, in a dose (10–200 μM)- and time-dependent manner (Fig. 3) , with TM cells exhibiting a stronger response than CB cells. Because 100- and 200-μM drug–induced changes in cell morphology and cytoskeletal aspects were found to be identical, data obtained with 200 μM are not shown. Blebbistatin-induced changes in TM cell morphology, actin cytoskeletal organization, and cell adhesions were confirmed in three independent experiments. 

Blebbistatin-induced changes in cell shape, actin cytoskeletal organization, and focal adhesions were found to be reversible on washout of drug from the cell culture medium. Cells treated with 100 μM drug for 5 hours showed complete restoration of normal cell shape, actin cytoskeletal organization, and focal adhesions 24 hours after drug removal from the culture medium (Fig. 4) . Cells treated with 200 μM blebbistatin, in contrast, required more than 48 hours for recovery of normal morphology after removal of the drug from the cell culture medium (data not shown). 

To evaluate viability and cytotoxicity in drug-treated TM and CB cells, we treated porcine TM and CB cells initially with blebbistatin (100 or 200 μM) for 1 to 5 hours in the presence of serum. TM cells treated with 100 μM drug for 5 hours exhibited no detectable cytotoxicity, and all cells were found to be viable based on fluorescein diacetate staining. A small number of TM cells treated with 200 μM drug for 3, 4, or 5 hours began to exhibit positive staining for propidium iodide, with the percentages ranging from 7% to 14%. TM cells treated for 2 hours with 200 μM drug exhibited no detectable positivity for propidium iodide staining (data not shown). Similarly, CB cells treated with 100 or 200 μM drug for 5 hours showed positive staining for propidium iodide in 5% and 18% of cells, respectively. Cells maintained in culture medium alone (control) did not exhibit detectable propidium iodide staining. These studies were conducted in triplicate, with all three sets of cells demonstrating consistent responses. 

Effects of Blebbistatin on MLC Phosphorylation in Porcine TM Cells

To explore the effects of blebbistatin on MLC phosphorylation status in TM cells, confluent cultures of porcine TM cells were treated with 25, 50, or 100 μM blebbistatin for 2 hours, and equal amounts of total protein were analyzed for changes in MLC phosphorylation by Western blot analysis. Data from two independent experiments revealed no differences in MLC phosphorylation status between control and drug-treated samples. Figure 5depicts representative data of blebbistatin’s effects on MLC phosphorylation in TM cells. 

Effects of Blebbistatin on Aqueous Outflow Facility in Enucleated Porcine Eyes

Freshly enucleated porcine eyes obtained from a local abattoir were perfused with blebbistatin (100 or 200 μM) at a constant pressure of 15 mm Hg, after the baseline outflow facility was established with PBS buffer containing d-glucose at 25°C (Fig. 6) . Basal rates of outflow facility (in microliters/minute per mm Hg) determined after 1 hour of perfusion were 0.269 ± 0.040 (mean ± SE, n = 6) and 0.414 ± 0.062 (n = 7) in the control groups, and 0.349 ± 0.046 (n = 6) and 0.353 ± 0.059 (n = 7) in the treatment groups that were subsequently perfused with 100 or 200 μM blebbistatin, respectively. After baseline facility was established, test samples were perfused with blebbistatin at room temperature, for 5 hours at 15 mm Hg. Aqueous outflow facility was observed to increase significantly (P < 0.05) after 1 hour of perfusion with either 100 or 200 μM blebbistatin (19% and 22% increases over the sham-treated control, respectively, P < 0.05). Outflow facility continued to increase with length of drug perfusion, being 32% and 45% higher than the corresponding sham-treated control (P < 0.01, Fig. 6 ) after 4 hours of perfusion with 100 or 200 μM blebbistatin, respectively. Mean values (mean ± SE) of outflow facility (in microliters/minute per mm Hg) were 0.526 ± 0.067 and 0.569 ± 0.085 in the 100- and 200-μM drug-perfused eyes, respectively, and 0.365 ± 0.033 and 0.503 ± 0.076 in the sham-treated control groups. These data reveal a dose- and time-dependent increase in outflow facility in response to perfusion with blebbistatin. Sham-treated eyes showed an increase of 21% from baseline facility, indicative of a washout response in porcine eyes. 

In addition to 100 and 200 μM blebbistatin, we evaluated the effects of lower concentrations of drug (25 μM) on aqueous outflow facility. Perfusion with 25 μM did not yield a significant difference in aqueous humor outflow facility, based on the mean level in six individual samples. The facility at 5 hours after perfusion was 0.277 ± 0.048 (n = 6) and 0.264 ± 0.019 (n = 6) in drug-perfused and sham-treated samples, respectively. The baseline facility in sham- and drug-treated samples was 0.191 ± 0.016 and 0.197 ± 0.035, respectively. The percentage of change in aqueous outflow facility from the baseline facility was not significantly different between drug-perfused and sham-treated eyes (data not shown). 

Histologic Integrity of Aqueous Outflow Pathway in Blebbistatin-Perfused Porcine Eyes

The integrity of the inner wall of aqueous plexi and trabecular meshwork in blebbistatin (200 μM for 5 hours)- and sham-treated eyes was analyzed by light and transmission electron microscopy. Different quadrants obtained from two drug- and sham-treated eyes revealed a continuous and intact inner wall of aqueous plexi (structurally equivalent to Schlemm’s canal in human eyes). Figure 7depicts the integrity of the aqueous outflow pathway in sham- and drug-treated specimens, respectively, and the left and right panels in these figures illustrate the histologic integrity of different regions of outflow at different magnifications. A small numbers of giant vacuoles (GVs) were found in the inner wall of aqueous plexi in both control and drug-treated eyes, with no apparent differences in number or size between the two groups. Similarly, the juxtacanicular area (JCT) or the subendothelial region of aqueous plexi was found to be intact in both sham- and drug-treated eyes (Fig. 7) . Both the inner (close to the JCT) and outer TM in drug-perfused eyes showed no detectable differences in geometry or cell morphology (Fig. 7 , arrowheads) compared with the TM in sham-treated eyes (Figs. 7A 7B , bottom panels). A second set of eyes also revealed identical histologic findings, with no distinctions being noted between control and drug-perfused specimens (data not shown). 

Discussion

The regulation of contractile activity of the TM and ciliary body by both calcium-dependent and -independent pathways, has been implicated in aqueous humor outflow facility. ^{6 18 25 28} Based on the use of various pharmacological modulators of contractile function of ciliary and TM cells, inhibitors of TM tissue contractility have been suggested to be of potential therapeutic significance in lowering intraocular pressure in patients with glaucoma. ^{17 18 25 28 29 34} Members of the myosin II family, which are adenosine triphosphate (ATP)-driven molecular motors, play critical role(s) in various cellular processes, including muscle contraction, cytokinesis, cortical tension maintenance, and neurite outgrowth and retraction. ^{22 43} To gain a better understanding of the significance of the contractile function of TM and ciliary muscle for aqueous outflow and to identify novel pharmacological avenues to lower IOP, in this study we examined the effects of a recently characterized novel small molecule inhibitor of myosin II, blebbistatin, ³⁷ on aqueous humor outflow facility in enucleated porcine eyes. The results confirm the expression of nonmuscle myosin IIA and IIB isoforms in TM and ciliary body cells and demonstrate that perfusion of enucleated porcine eyes with blebbistatin leads to significant increases in aqueous outflow facility. Furthermore, this response to blebbistatin was associated with changes in cell morphology in TM and CB cells, decreased actin stress fibers, focal adhesions, and adherens junctions in a cell culture model. 

Blebbistatin was recently characterized as a small-molecule inhibitor of myosin II ATPase activity. ^{37 39} It is a potent inhibitor of skeletal muscle and nonmuscle myosin II isoforms, although it has little or no effect on smooth muscle myosin II and myosins from classes I, V, and X. ³⁹ Because of its selectivity and high affinity for class II myosins, blebbistatin is regarded as an important pharmacological tool in exploring the role of myosin II ATPase in cells and tissues. ³⁷ We therefore used blebbistatin to evaluate the specific role played by myosin II in maintenance of cell morphology and in actomyosin organization, cell adhesions and cell–cell junctions in TM and CB cells. Treatment of primary cultures of both porcine TM and CB cells with blebbistatin (10–200 μM for 2 hours) led to reversible alterations in cell morphology and decreases in actin stress fibers, focal adhesions and adherens junctions, without detectable cytotoxic effects (Fig. 3) . The concentration of blebbistatin used in our studies (50–100 μM) has been reported to inhibit predominantly nonmuscle and skeletal muscle myosin II in vitro. ^{37 39 44 45} The effects of blebbistatin on TM cell morphology and actomyosin cytoskeletal organization observed in this study were consistent with the effects of the inhibitors of Rho kinase, protein kinase C, and MLCK on TM cells documented earlier. ^{17 27 33} However, unlike these inhibitors, which mediate their effects by inhibiting myosin II activity through decreased MLC phosphorylation, ^{17 27 33 34} blebbistatin (50–100 μM) mediates its effects without affecting the phosphorylation status of MLC in TM and CB cells (Fig. 4) , confirming its specificity to myosin II ATPase activity. The TM and ciliary body tissues have been reported to exhibit distinct contractile responses when treated with external factors. ¹⁸ In this study, however, both the TM and CB treated with blebbistatin revealed similar responses in cell morphology, actin cytoskeletal reorganization, and cell adhesions. The concentration of drug needed for CB cells was relatively higher than the effective concentration required for response generation in TM cells (Fig. 3) . 

Perfusion of enucleated porcine eyes with blebbistatin (100 or 200 μM) for 5 hours at constant pressure (15 mm Hg) led to a significant dose- and time-dependent increase in aqueous outflow facility (Fig. 6) . Aqueous outflow facility began to exhibit a significant increase within an hour of drug perfusion and continued to increase in response to 100 μM drug. Eyes perfused with 200 μM drug revealed a similar and significant increase in aqueous outflow facility, evident after 1 hour of drug perfusion and continuing up to 4 hours, after which facility was noted to decline. In cell culture studies, although no cytotoxicity was observed at the 100-μM drug concentration over the 5-hour period, 200 μM blebbistatin appeared to exert some cytotoxic effects after 3 hours of incubation. Histologic analysis of the aqueous outflow pathway in drug-perfused eyes, however, revealed an intact and continuous lining of inner wall of aqueous plexi (structurally equivalent to Schlemm’s canal in primates). Although blebbistatin treatment induced changes in cell shape and decreased actin stress fibers and cell rounding and detachment in primary TM cell cultures, TM cells on the trabecular beams appeared normal and show no detectable changes in morphology in drug-perfused eyes. It is postulated that with inhibition of myosin II ATPase activity by blebbistatin, the tissue and cells of the aqueous outflow pathway undergo relaxation due to reduced interaction between actin and myosin, leading to changes in cell morphology of outflow pathway and altered geometry of the outflow pathway. However, as shown in Figure 7 , the blebbistatin-induced increase in the aqueous outflow facility was not associated with obvious differences in the integrity of aqueous outflow or the morphology of TM tissue. It is also conceivable that the blebbistatin-induced morphologic and histologic changes in vivo are subtle enough to be undetectable by the techniques used in our study. Moreover, it is recognized that cells in culture usually manifest more pronounced morphologic and cytoskeletal changes than their counterparts in an intact tissue environment (Fig. 3) . Therefore, although 25 μM blebbistatin causes definite changes in TM cell morphology and actin cytoskeletal organization under cell culture conditions (Fig. 3) , we did not observe significant differences in aqueous outflow facility at this concentration, in perfusion studies. A plausible reason for this discrepancy could be that cells in the tissue are associated with various components of the extracellular matrix, whereas cells in plastic dishes exist in a simpler environment. Therefore, cells in the cell culture model may be intrinsically more sensitive to the drug and thus exhibit more pronounced changes that are easily detectable. 

It has been proposed that alterations in cell–cell and cell–ECM interactions, and cell morphology modulate the permeability barrier function of inner wall of SC and the geometry of TM, thereby potentially accounting for the increased aqueous humor outflow facility. ^{8 16 17} The lack of clear experimental evidence of such morphologic or histologic changes in this study and others, ^{28 46} however, emphasizes the need for considering and exploring alternate hypotheses. For example, ECM organization and turnover at the JCT may also be influenced by the changes in contractile characteristics and actin cytoskeletal organization in cells of the outflow pathway, ^{47 48} and such changes may be involved in regulating aqueous outflow facility. 

An intriguing aspect of blebbistatin is that it has been shown to be a very poor inhibitor of purified smooth muscle myosin II, even at a concentration of 100 μM. ³⁹ However, 100 μM blebbistatin was noted to induce a significant increase in aqueous outflow facility in the perfusion experiments conducted in our study (Fig. 6) . Furthermore, because concentrations of 10 to 25 μM blebbistatin were observed to have effects in cultured TM cells, we speculate that the effects of blebbistatin on outflow facility and on TM cell cytoskeletal changes are not related to smooth muscle myosin II (Fig. 3A) . The contractile function of TM smooth muscle has been thought to have a critical role in the regulation of aqueous humor outflow, ¹⁸ and TM tissue has been shown to possess smooth-muscle–like characteristics and to express smooth-muscle–specific proteins, including α-smooth muscle actin, myosin, and CPI-17. ^{18 28 49 50} Although we did not determine the expression of smooth muscle myosin II in TM or CB in this study, it is conceivable that the blebbistatin-induced alterations in aqueous outflow facility observed in this study are solely or predominantly associated with cells of a nonmuscle phenotype. In support of this notion, RT-PCR-based analysis confirmed expression of nonmuscle myosin IIA and IIB in human TM and CB cells (Fig. 2) . It becomes important then to consider the role and significance of nonmuscle cells and their contractile characteristics in the aqueous humor outflow pathway. This may provide further insight into our understanding of TM contractile function and its regulation, given how contractile activity of smooth muscle and nonmuscle cells is known to be regulated differentially. ^{22 51}  

In addition, it is important to consider that myosin II is a functional target for the Rho kinase inhibitor–induced increase in aqueous humor outflow facility in different species. However, the Rho kinase inhibitor–induced effects on outflow facility have been found to be greater than blebbistatin-induced changes. ^{17 28} A possible explanation for the stronger influence of Rho kinase inhibitors on outflow facility is that, unlike blebbistatin, which preferentially inhibits nonmuscle myosin II, inhibitors of Rho kinase inhibit both smooth muscle and nonmuscle myosin II activity by regulating myosin phosphatase activity, ^{20 22 24} indicating that the inhibition of different myosin IIs, including smooth muscle and nonmuscle, in the outflow pathway may have a stronger effect on aqueous outflow facility than the inhibition of either one alone. 

In conclusion, in this study, myosin II played a critical role in modulation of aqueous outflow facility and inhibition of myosin II by blebbistatin increased aqueous outflow facility. Thus, the direct inhibition of myosin II in the aqueous outflow pathway may be of therapeutic significance in lowering intraocular pressure in patients with glaucoma. 

 Supported by National Eye Institute Grants EY013573, EY12201 (PVR), and P-30 EY05722, and funding from Research to Prevent Blindness.

 Submitted for publication February 8, 2005; revised May 12, July 5, and July 18, 2005; accepted August 30, 2005.

 Disclosure: M. Zhang, None; P.V. Rao, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Structure of (±)-blebbistatin. Reprinted with permission from Straight AF, Cheung A, Limouze J, et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science. 2003;299:1743–1747. © The American Association for the Advancement of Science.

Table 1.

 

Human Nonmuscle Myosin IIA, IIB, and IIC Oligonucleotide Primer Pairs Used in RT-PCR Analysis

Gene	Primer	Product Size (bp)
MYH9 (myosin IIA)	5′-GAAGGTCATCCAGTATCTGGCG	355
	5′-ACAGGAAGCGGTATTTGTTGTACG
MYH10 (myosin IIB)	5′-AGAAGGGCATGTTTCGTACCG	236
	5′-TGAATTCCTGGAAAACTATTCGGTTAG
MYH14 (myosin IIC)	5′-AAGCCATTGTGGAGATGTACCG	386
	5′-GGTAGGTCTCAATGTTGGCGC

Figure 2.

 

Expression of nonmuscle myosin II in human and porcine TM tissue. Expression of nonmuscle myosin II isoforms was determined in human TM and CB cells by RT-PCR analyses, using sequence-specific oligonucleotide primers (A). Both human TM and CB cells were confirmed to express nonmuscle myosin IIA and IIB by sequencing the PCR products. Only a myosin IIB-specific PCR product was amplified with cDNA derived from human TM tissue. TM cells derived from different donor eyes revealed similar expression profiles of nonmuscle myosin II. Because a search of GenBank data and other sequence databases did not identify a porcine-specific sequence for myosin II, an attempt was made to amplifying myosin IIA and IIB from the porcine TM tissues using oligonucleotide primers directed against the human species (Table 1) . RT-PCR-generated cDNA libraries (two independent samples) from porcine TM tissue yielded an easily detectable DNA product for myosin IIB (B). The DNA product was sequenced to confirm identity, and BLAST sequence identity analysis revealed the highest sequence homology with that of mouse myosin IIB. In contrast, human myosin IIA oligonucleotide primers did not yield any DNA product, indicating possibly significant sequence differences in the myosin IIA gene between humans and pigs. Marker: 100-bp molecular measure.

Figure 3.

 

Blebbistatin-induced changes in actin stress fibers, focal adhesions, and adherens junctions in porcine TM (A) and CB (B) cells. Confluent cultures of TM and CB cells were treated with 10 to 100 μM blebbistatin for 2 hours at 37°C, in the presence of 10% FBS. Cells were subsequently stained for actin (with rhodamine-conjugated phalloidin), focal adhesions (with anti-vinculin antibody), or adherens junctions (with anti-β-catenin antibody). In both porcine TM and CB cells, blebbistatin induced changes in cell morphology and dose-dependent decreases in actin stress fibers, focal adhesions (arrows), and adherens junctions (arrows). Magnification, ×400.

Figure 4.

 

Reversibility of blebbistatin-induced changes in TM cell morphology and actin cytoskeletal organization changes on drug withdrawal. Confluent cultures of porcine TM cells were treated with 100 μM blebbistatin for 5 hours at 37°C in the presence of 10% FBS, after which, the drug was withdrawn by rinsing (minimum of three times) the cells with culture medium containing no drug. After 24 hours of drug removal, cells were fixed and stained for actin (with rhodamine-phalloidin) and focal adhesions (with anti-vinculin antibody). Blebbistatin-induced changes in TM cell shape and actin cytoskeletal organization, and focal adhesions showed complete reversal within 24 hours of drug withdrawal from the culture medium. These results were confirmed in three independent experiments. Magnification, ×400.

Figure 5.

 

Blebbistatin-induced changes in myosin light chain phosphorylation in porcine TM cells. Confluent cultures of porcine TM cells treated with 25, 50, or 100 μM blebbistatin for 2 hours exhibited no effects on MLC phosphorylation status, as determined by Western blot analysis using a phospho-MLC-specific polyclonal antibody. Immunoblots showed a single immunopositive band corresponding to the molecular mass of MLC. Equal amounts of total protein from control and drug-treated samples were used in this analysis, and actin was also probed in these samples by Western blot analysis, to confirm loading equivalency. Similar data were recorded in two independent experiments.

Figure 6.

 

Blebbistatin increased aqueous humor outflow facility in enucleated porcine eyes. Contralateral paired eyes were perfused with PBS containing 5.5 mM glucose under a constant pressure of 15 mm Hg at 25°C. After initial baseline outflow facility was established, eyes were perfused with 100 or 200 μM blebbistatin for 5 hours, with outflow facility being monitored at hourly intervals. Both control and test group eyes exhibited similar basal outflow facility (see the Results section). Outflow facility was observed to increase significantly after 1 hour of blebbistatin perfusion (using either 100 or 200 μM drug) and continue to increase progressively and significantly over the 5-hour perfusion period with 100 μM drug. Outflow facility was noted to increase significantly for the first 4 hours of perfusion with 200 μM drug, but then declined slightly by the fifth hour of perfusion. Drug-induced changes in aqueous outflow facility are calculated as the percentage change in outflow facility from baseline. *P < 0.05, **P < 0.01, and ***P < 0.001: significant differences in aqueous humor outflow facility between sham- and drug-perfused eyes.

Figure 7.

 

Effects of blebbistatin perfusion on the integrity of aqueous plexi and trabecular meshwork of enucleated porcine eyes. Enucleated porcine eyes perfused with blebbistatin (200 μM) for 5 hours at 25°C were fixed for histologic examination by light and electron microscopy. The integrity of the inner wall of aqueous plexi (indicated with arrows) in drug-perfused eyes (200 μM; B) was found to be intact, and TM morphology appeared to be similar to that of the control (A). No apparent difference was noted in either the size or number of GVs in the inner wall of aqueous plexi of drug-perfused eyes, compared with the corresponding control. Different tissue quadrants obtained from two drug-perfused and control eyes showed similar data. (A, B) Top and bottom panels focus on the integrity of aqueous plexi (structurally equivalent to human SC) and TM, respectively. Left and right panels show different magnifications of the same specimen. Arrowheads: cells on the TM beams. Magnification: (left) ×1200; (right) ×2000.

The authors thank David Epstein for helpful discussion during these studies, Keith Burridge (University of North Carolina, Chapel Hill, NC) for introducing blebbistatin; and Peifeng Deng and Wen Xiu Zhang for help in perfusion studies and histologic analysis, respectively.