The role of calcium-independent phospholipase A2γ in modulation of aqueous humor drainage and Ca2+sensitization of trabecular meshwork contraction

Abstract

The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A2(iPLA2), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA2γ specific) and (S)-bromoenol lactone (S-BEL, iPLA2β specific). Immunohistochemical analysis revealed intense staining for both iPLA2β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA2γ by R-BEL or small interfering RNA-mediated silencing of iPLA2γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA2γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA2γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.