Enzymatic and metabolic products of a distinct phospholipase in the retina: Towards the development of cell-based assays for a high-throughput screen

Authors

• JL Bullock
• SP Becerra

Abstract

Retinitis pigmentosa and macular degeneration are caused by degeneration of photoreceptor cells, which progressively leads to blindness. Our lab has identified pigment epithelium-derived factor receptor (PEDF-R) as a cell surface receptor for PEDF, a protective protein of the eye. Upon binding, PEDF enhances the phospholipase activity of PEDF-R, which is critical for the retinal survival effects mediated by PEDF. Fatty acids and lysophoshphatidylcholine (LPC) or lysophosphatidic acid (LPA) molecules are products of the phospholipase activity. Fatty acids can then be catabolized via ß-oxidation, which leads to the production of the ketone body ß-hydroxybutyrate (B-HB). We sought to evaluate the levels of LPC, LPA, and B-HB in retinal R28 precursor cells in the absence or presence of PEDF. After 24 hours of serum starvation, LPC levels in conditioned media of PEDF treated cells were comparable to LPC levels in untreated cells, while LPA levels decreased with PEDF treatment. LPA levels of culturing media from PEDF treated cells were similar to those from cells cultured with serum-containing medium. There were no significant differences between intracellular LPA levels of cells treated with PEDF and cells cultured in serum-free medium. B-HB increased in PEDF treated cells over time. Our findings show that the products of PEDF-R activity can be measured in conditioned medium from retinal cells in culture. The results can be used to develop a high-throughput assay for the identification of PEDF-R enhancers, leading towards the discovery of potential retinal degeneration therapeutics.

Scientific Focus Area: Molecular Biology and Biochemistry

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