Effect of H-7 on cultured human trabecular meshwork cells.
OBJECTIVE
To determine the effect of the serine-threonine kinase inhibitor H-7, which blocks actomyosin contractility and increases outflow facility in live monkeys, on morphology, cytoskeleton, and cellular adhesions of human trabecular meshwork (HTM) cells in culture.


METHODS
Cultured HTM cells were videographically recorded and evaluated before and after exposure to H-7 at different concentrations. The subcellular distribution of the actin-based cytoskeleton and associated anchor proteins including vinculin, paxillin, and beta-catenin, as well as phosphotyrosine-containing proteins were evaluated by fluorescence immunocytochemistry and digital fluorescence microscopy.


RESULTS
H-7 induced pronounced but reversible HTM cell thickening toward the cell center and deterioration of the actin cytoskeletal network. Cell-extracellular matrix (ECM) and cell-cell adhesions were also affected, but the beta-catenin-rich, vinculin-containing adherens junctions were clearly more resistant than focal contacts. Phosphotyrosine labeling in focal contacts was highly sensitive to H-7.


CONCLUSIONS
H-7 induces alterations in cell shape, actin cytoskeleton, and associated focal adhesions in cultured HTM cells, which may be responsible for the effects of H-7 on outflow facility in live monkey eyes.
Liu
X
X
Department of Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI 53792, USA. xliu5@facstaff.wisc.edu
Cai
S
S
Glasser
A
A
Volberg
T
T
Polansky
J R
JR
Fauss
D J
DJ
Brandt
C R
CR
Geiger
B
B
Kaufman
P L
PL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology*; D000199:Actins / Q000378:metabolism; D002448:Cell Adhesion; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015534:Trans-Activators; D016596:Vinculin / Q000378:metabolism; D051176:beta Catenin
D000199:Actins; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D015534:Trans-Activators; D051176:beta Catenin; D016596:Vinculin; D019000:Phosphotyrosine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases