Acto-myosin drug effects and aqueous outflow function.
OBJECTIVE
Previous studies have identified the cytoskeletal proteins actin and tubulin as potential cellular targets in the trabecular meshwork for novel glaucoma therapy. The authors and others have hypothesized that acto-myosin interactions may be important for outflow function. The current study was conducted to evaluate 2,3-butanedione 2-monoxime (BDM), a compound that interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction; 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a proposed myosin light-chain kinase inhibitor; and the direct actin disrupter, latrunculin B, in an outflow pathway cell culture and perfused excised eye model system.


METHODS
Freshly enucleated porcine eyes were perfused using the constant-pressure method at 15 mm Hg and 25 degrees C. Human trabecular meshwork (HTM) cells and Schlemm's canal (SC) cells were grown in culture, treated with BDM, H-7, and latrunculin B, and then fixed, stained for beta-tubulin and filamentous actin, and observed by epifluorescence.


RESULTS
Twenty millimolar BDM, 100 microM H-7, and 1 microM latrunculin B increased outflow facility 36%, 63%, and 72%, respectively, compared to sham-treated controls, 13%, 15%, and 4% (n=7, 8, and 8; P=0.01, 0.0001, and 0.0002), respectively. In cultured HTM and SC cells, 100 microM H-7 caused a rapid loss of filamentous actin staining but did not produce a change in cell shape or cell- cell attachment. In contrast, 20 mM BDM induced a loss of cell- cell attachment and a change in cell shape that was associated with a 50% to 60% loss of filamentous actin staining, often in a distinct stick-and-ball pattern. Latrunculin B caused a severe loss of actin staining and cell shape changes. No drug altered beta-tubulin staining.


CONCLUSIONS
Interference with myosin function can cause a secondary loss of actin organizational structure. Our study indicates that myosin, perhaps through its various phosphorylation reactions, may have a potential regulatory role in outflow function.
Epstein
D L
DL
Department of Ophthalmology, Duke University Medical Center, Duke University Eye Center, Durham, North Carolina 27710, USA.
Rowlette
L L
LL
Roberts
B C
BC
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology*; D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000187:drug effects* / Q000378:metabolism; D013552:Swine; D013844:Thiazoles / Q000494:pharmacology*; D053778:Thiazolidines; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D013844:Thiazoles; D053778:Thiazolidines; C004717:diacetylmonoxime; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D009218:Myosins; D003931:Diacetyl; C037068:latrunculin B