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

Abstract

Pattabiraman PP, Lih FB, Tomer KB, Rao PV. The role of calcium-independent phospholipase A 2␥ in modulation of aqueous humor drainage and Ca 2ϩ sensitization of trabecular meshwork contraction. Am J Physiol Cell Physiol 302: C979 -C991, 2012. First published January 11, 2012; doi:10.1152/ajpcell.00396.2011.-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 A 2 (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, iPLA 2␥ specific) and (S)-bromoenol lactone (S-BEL, iPLA 2␤ specific). Immunohistochemical analysis revealed intense staining for both iPLA 2␤ and ␥ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA 2␥ by R-BEL or small interfering RNA-mediated silencing of iPLA 2␥ 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 iPLA 2 ␥ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA 2␥ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways. Rho GTPase; arachidonic acid; glaucoma GLAUCOMA IS AN OPTIC NEUROPATHY that leads to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma and is associated with elevated intraocular pressure (IOP). Elevated IOP is a major risk factor for glaucoma (41,54), and arises mainly due to an abnormally increased resistance to the drainage of aqueous humor (AH) through the conventional outflow pathway comprising of trabecular meshwork (TM) and the Schlemm's canal (SC) (28,54). Pharmacological interventions to treat glaucoma have been classically aimed at lowering elevated IOP by either decreasing secretion of AH by the ciliary body, or augmenting AH drainage by the uveoscleral and conventional pathways (29). Recently, significant advances have been made towards identifying the role of cellular processes involved in regulating AH outflow dynamics and the potential of s...

Complement-mediated Activation of Calcium-independent Phospholipase A2γ

“…PLA 2 Assay-PLA 2 activity was measured in COS-1 cell extracts using a PLA 2 activity assay kit according to the manufacturer's instructions and as described previously (42). In this assay, hydrolysis of arachidonoyl thiophosphatidylcholine at the sn-2 position by PLA 2 releases a free thiol that is detected by 5,5Ј-dithio-bis-2-nitrobenzoic acid.…”

Bioactive Lysophospholipids: Role in Regulation of Aqueous Humor Outflow and Intraocular Pressure in the Context of Pathobiology and Therapy of Glaucoma

“…42,44,80 In addition, enzymes generating these lysolipids were found to influence AH outflow and IOP. 43,81 There is also some evidence of dysregulation of LPA production in the AH and optic nerve head of glaucoma patients. 43,82,83 In addition, these bioactive lipids have been shown to play a critical role in the expression of ECM and a-smooth muscle actin.…”

“…111 Very recently, we not only confirmed the distribution pattern of 2 isoforms of calcium-independent phospholipaseA2 (iPLA2b and iPLA2g) in human TM and cultured TM cells, but also established their role in regulation of AH outflow facility. 81 Using isoform-specific small molecular inhibitors of iPLA2 (S-Bromoenol lactone) and (R-Bromoenol lactone), it has been demonstrated that iPLA2g plays a significant role in the regulation of TM cell contractile properties, cell shape, and cell adhesive interactions by controlling MLC phosphorylation. 81 These changes appear to be mediated by the signaling activities of Rho GTPase, PKC, and arachidonic acid in TM cells.…”

“…81 Using isoform-specific small molecular inhibitors of iPLA2 (S-Bromoenol lactone) and (R-Bromoenol lactone), it has been demonstrated that iPLA2g plays a significant role in the regulation of TM cell contractile properties, cell shape, and cell adhesive interactions by controlling MLC phosphorylation. 81 These changes appear to be mediated by the signaling activities of Rho GTPase, PKC, and arachidonic acid in TM cells. 81 Importantly, the inhibition of iPLA2g but not iPLA2b appears to suppress Rho GTPase activation and decrease the levels of total arachidonic acid in TM cells.…”

“…81 These changes appear to be mediated by the signaling activities of Rho GTPase, PKC, and arachidonic acid in TM cells. 81 Importantly, the inhibition of iPLA2g but not iPLA2b appears to suppress Rho GTPase activation and decrease the levels of total arachidonic acid in TM cells. 81 Significantly, the inhibition of iPLA2g in perfused enucleated porcine eyes led to an increased AH outflow facility in association with increased TM relaxation.…”