Cytochrome P450-mediated prostaglandin [formula omitted] hydroxylase activities in porcine ciliary body epithelial cells

Abstract

The ocular hypotensive effect of topically applied prostaglandins (PGs) is well documented. Although PGs introduced in the posterior chamber accumulate in the anterior tissues (e.g. iris/ciliary complex), little is known about the metabolism of PGs by these tissues. We have recently found that non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells are readily separated from porcine ciliary body and cytochrome P450-dependent xenobiotic metabolism is considerably higher in NPE cells than in PE cells. We have therefore investigated in this study the cytochrome P450-mediated PG ω ω-1 hydroxylase activities of porcine ciliary epithelial cells. The NPE cells show about three times higher activities than do PE cells; the NPE cells, in fact, demonstrate the highest PG ω ω-1 hydroxylase activities among different ocular tissues. Both ω and ω-1 hydroxylases show broad substrate specificities and hydroxylate PGA1, A2, E1, E2, and lauric acid. The ω ω-1 hydroxylase activities of NPE and PE, as determined with PGA2 and lauric acid as substrates, are enhanced or induced by treatment of primary cultures of the individual epithelial cells with clofibrate, both activities reaching maximum levels within 48 hr of induction. The induced activities are inhibited almost completely by cycloheximide and actinomycin D. The ω ω-1 hydroxylase activities of both NPE and PE cells require NADPH and molecular oxygen, are associated with the microsomal fraction, respond to inducers such as clofibrate, and are inhibited by metyrapone and SKF525 A (inhibitors of P450 enzymes). These results support the suggestion that PG ω ω-1 hydroxylations by NPE and PE are cytochrome P450-mediated reactions. The primary association of P450-mediated ω ω-1 hydroxylase activities with NPE cells supports the contention that ciliary NPE cells play an important role in the metabolism of xenobiotics as well as endogenous substances (autacoids) in the anterior part of the eye.