Blebbistatin, a novel inhibitor of myosin II ATPase activity, increases aqueous humor outflow facility in perfused enucleated porcine eyes.
OBJECTIVE
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 microM)-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 microM 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.
Zhang
Min
M
Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Rao
P Vasantha
PV
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D002924:Ciliary Body / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D015353:Eye Enucleation; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D024507:Nonmuscle Myosin Type IIA / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010477:Perfusion; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D006576:Heterocyclic Compounds, 4 or More Rings; C472645:blebbistatin; D000251:Adenosine Triphosphatases; D024507:Nonmuscle Myosin Type IIA; D024501:Nonmuscle Myosin Type IIB