Effects of pharmacologic inhibition of protein geranylgeranyltransferase type I on aqueous humor outflow through the trabecular meshwork.
OBJECTIVE
To determine the effects of inhibition of protein geranylgeranyltransferase type I (GGTase-I), which isoprenylates so-called CaaX proteins, including the GTP-binding proteins such as Rho GTPases and the betagamma subunits of heterotrimeric G-proteins, on aqueous humor outflow and trabecular meshwork cytoskeletal integrity.


METHODS
A selective small molecular inhibitor of GGTase-I, GGTI-DU40, was tested in this study to investigate its effects on actin cytoskeletal integrity, cell adhesions, cell-cell junctions, myosin II phosphosphorylation, and membrane localization of GTP-binding proteins in trabecular meshwork (TM) cells, using immunofluorescence detection and immunoblotting analysis. The effects of GGTI-DU40 on aqueous humor outflow were determined using organ-cultured, perfused anterior segments of porcine eyes.


RESULTS
In the TM cell lysates, GGTI-DU40 was confirmed to inhibit GGTase-I activity in a dose-dependent manner. TM cells treated with GGTI-DU40 displayed dose-dependent changes in cell morphology and reversible decreases in actin stress fibers, focal adhesions, and adherens junctions. Myosin light chain phosphorylation was decreased significantly, and membrane localization of isoprenylated small GTPases and Gbetagamma was impaired in drug-treated TM cells. Aqueous outflow facility was increased significantly in eyes perfused with GGTI-DU40.


CONCLUSIONS
These data demonstrate that inhibition of geranylgeranyl isoprenylation of CaaX proteins in the aqueous outflow pathway increases aqueous humor outflow, possibly through altered cell adhesive interactions and actin cytoskeletal organization in cells of the outflow pathway. This study indicates that the GGTase-I enzyme is a promising molecular target for lowering increased ocular pressure in glaucoma patients.
Rao
P Vasantha
PV
Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina 27710, USA. rao00011@mc.duke.edu
Peterson
Yuri K
YK
Inoue
Toshihiro
T
Casey
Patrick J
PJ
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D022001:Focal Adhesions / Q000378:metabolism; D007365:Intercellular Junctions / Q000187:drug effects; D008858:Microscopy, Phase-Contrast; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D024462:Myosin Type II / Q000378:metabolism; D009924:Organ Culture Techniques; D010766:Phosphorylation; D017368:Protein Prenylation; D011720:Pyrazoles / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013997:Time Factors; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000201:enzymology
D000199:Actins; D004791:Enzyme Inhibitors; C530102:GGTI-DU40; D018994:Myosin Light Chains; D011720:Pyrazoles; D011725:Pyridines; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D024462:Myosin Type II; D020559:Monomeric GTP-Binding Proteins