April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Inhibition of Calcium-Independent Phospholipase A2 Increases Aqueous Humor Drainage Through the Conventional Pathway
Author Affiliations & Notes
  • Padmanabhan P. Pattabiraman
    Ophthalmology, Duke University Medical Center, Durham, North Carolina
  • Vasanth Rao
    Ophthal & Pharmacology, Duke University Eye Center, Durham, North Carolina
  • Footnotes
    Commercial Relationships  Padmanabhan P. Pattabiraman, None; Vasanth Rao, None
  • Footnotes
    Support  R01 EY018590
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 6619. doi:
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      Padmanabhan P. Pattabiraman, Vasanth Rao; Inhibition of Calcium-Independent Phospholipase A2 Increases Aqueous Humor Drainage Through the Conventional Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6619.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To determine the role of calcium-independent phospholipase A2 (iPLA2), which is known to control eicosanoid synthesis and tissue contractile properties, in regulation of aqueous humor outflow.

Methods: : iPLA2 mRNA expression and protein levels were determined by RT-PCR and immunoblot analyses, respectively, in cultured trabecular meshwork (TM) cells derived from human and porcine eyes. Distribution of iPLA2 isoforms in the outflow pathway tissues was evaluated by immunohistochemical analysis. Using irreversible, chiral, mechanism-based isoform specific inhibitors of iPLA2, R-Bromoenol lactone (R-BEL, iPLA2γ specific) and S-Bromoenol lactone (S-BEL, iPLA2β specific), we determined the specific role(s) of iPLA2 isoforms in regulation of TM cell contractile and relaxation properties by biochemical analyses. S-BEL and R-BEL were perfused through enucleated porcine eyes, which were then evaluated for changes in aqueous outflow facility using a constant pressure perfusion system. Drug effects on the integrity of outflow pathway tissues were evaluated by light and transmission electron microscopy.

Results: : Both human and porcine TM cells were confirmed to express iPLA2 β and γ by RT-PCR and immunoblot analyses. Immunohistochemical analysis of iPLA2 β and γ showed an intense staining of both isoforms distributing throughout the TM, juxtacanalicular tissue and Schlemm’s canal of human eye. Inhibition of iPLA2γ by R-BEL (10 µM for 2 hours) induced dramatic changes in TM cell morphology (cell rounding), and decreased actin stress fibers, focal adhesions and myosin light chain phosphorylation. Aqueous humor outflow facility in the enucleated porcine eyes increased progressively and significantly (P<0.001, n=12) by 60% and 80% over the sham-treated control eyes, following R-BEL (25 µM) perfusion at 1hr and 5hr, respectively. This response was associated with a significant decrease in myosin light chain phosphorylation in the drug perfused TM tissue and increased formation of giant vacuoles in the innerwall of Schlemm’s canal. Contrarily, S-BEL did not influence either aqueous outflow facility or TM cell contractile properties.

Conclusions: : This is the first study to demonstrate a critical and isoform specific involvement of iPLA2 in regulation of aqueous humor outflow through the conventional pathway. Importantly, pharmacological inhibition of iPLA2 γ increases aqueous humor outflow facility through the trabecular pathway by affecting the tissue contractile characteristics and morphology.

Keywords: outflow: trabecular meshwork • eicosanoids • signal transduction: pharmacology/physiology 

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