High Affinity Binding Sites for 12(R)-Hydroxyeicosatrienoic Acid [12(R)-HETrE] in Microvessel Endothelial Cells

Abstract

12(R)-HETrE is an NADPH-dependent arachidonic acid-derived metabolite whose synthesis is induced several fold in inflamed corneal epithelium correlating with the development of the in situ inflammatory response, i.e., vasodilatation, PMN chemotaxis, endothelial cell mitogenesis, and neovascularization. Because this novel eicosanoid may serve as an endogenous mediator of the angiogenic response in the cornea during inflammation, we probed microvessel endothelial cells for a specific binding site which could possibly account for the mechanism by which this eicosanoid initiates changes in cellular activity. Binding of radioactive ligand [³H-12(R)-HETrE] was saturable with time and concentration. Scatchard analysis indicated a single, saturable binding site for 12(R)-HETrE with a B_max=24,700 sites/cell and an apparent K_d=0.043 nM. Thin layer chromatography analysis of cell-associated ligand revealed that esterification of 12(R)-HETrE was 2-7 fold less than unesterified, cell bound ligand. The concentrations of 12(R)-HETrE at which maximum biological activity has been observed, i.e., 0.1 nM, roughly corresponds to the K_d value, suggesting a functional link to this binding site. These studies begin to reveal a potential mechanism by which 12(R)-HETrE stimulates microvessel endothelial cells to invade the cornea leading to corneal neovascularization.