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Lindsey H. Millard, William D. Stamer, David F. Woodward, Kikuko Watanabe, Sanae Hagesawa-Ishii, Atsuyoshi Shimada; Ocular Distribution of the Enzyme Prostamide/Prostaglandin F Synthase. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1514.
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Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme belonging to the thioredoxin superfamily. It catalyses the formation of prostamide (PM)F2α and prostaglandin (PG)F2α by reducing the precursor endoperoxides PMH2 and PGH2. Since both PMF2α and PGF2α lower intraocular pressure, establishing the distribution of this key enzyme in ocular anterior segment tissues represents the first step in determining a role for this enzyme in regulating aqueous humor dynamics.
Using a specific rabbit polyclonal antibody (IgG) for PM/PGFS, ocular distribution was determined by Western blotting using ocular tissue homogenates and immunohistochemistry. For immunohistochemical studies, C57BL mice were fixed by systemic perfusion with Bouin’s fixative. Eyes were embedded in paraffin and cut into 6-µm-thick sections. Sections were incubated with the anti-PM/PGFS IgG and then with secondary antibody (EnVision+ System HRP-anti-rabbit [DAKO]). Negative controls involved pre-incubating the anti-PM/PGFS IgG with purified PM/PGFS. Immunoreactions were visualized using diaminobenzidine as chromogen. Subsequently, further preparation with H&E staining was performed to reveal tissue structure and orientation.
Western blot analysis identified the presence of PM/PGFS in the mouse eye. It had the correct size (21-23 kDa) for the purified enzyme. Immunohistochemical analysis revealed positive staining in the non-pigmented cells of the ciliary processes and the trabecular meshwork. Surprisingly, immunopositive results were obtained in the conjunctiva.
The presence of PM/PGFS in the trabecular meshwork indicates that the biosynthetic products of PM/PGFS may contribute to the regulation of pressure dependent aqueous humor outflow. A further influence on aqueous humor dynamics may occur at the secretion level, since the enzyme is also present in the ciliary processes.
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