Pharmacological and Molecular Biological (RT-PCR) Characterization of Functional TP Prostanoid Receptors in Immortalized Human Non-Pigmented Ciliary Epithelial Cells

Abstract

Immortalized human non-pigmented ciliary epithelial (NPE) cells (ODM-2) were shown to express the mRNA for the prostanoid TP _α but not the TP _β receptor using reverse transcription-polymerase chain reaction (RT-PCR). These TP _α receptors were coupled to phospholipase C (PLC) and, thus, promoted phosphoinositide (PI) turnover. TP receptor agonists yielded the following potencies (EC₅₀s) in the PI turnover assays: I-BOP = 8.2 ± 1.1 nM; carbocyclic TA₂ = 87.5 ± 25.3 nM; U-44069 = 1.16 ± 0.32 μM; U-46619 = 1.2 ± 0.2 μ M (n = 4-17). Agonists selective for other prostanoid receptor subtypes (e.g., fluprostenol and sulprostone) were inactive. The agonist effects of U-44619 and I-BOP were potently blocked, in an apparent non-competitive manner (k_i = 53.9 ± 12 nM; pA₂s = 7.6-7.8; pK_bs = 7.38), by the TP receptor-selective antagonist, SQ29,548, but were unaffected by other prostanoid receptor antagonists (e.g., AH6809, AL-8810). The PLC inhibitor (U73122) inhibited U-46619-induced PI turnover (IC₅₀ = 4.3 ± 0.6 μM). The functional potencies of the compounds stimulating or inhibiting the TP receptor-mediated PI turnover in the NPE cells correlated well with the TP receptor binding affinities of these compounds at human platelet TP receptors (r = 0.98). These studies have shown the presence of the mRNA for and the expression of functional TP _α receptors coupled to PLC in human NPE cells. The TP _α receptors on NPE cells may be responsible for inhibiting aqueous humor production and may help explain the intraocular pressure-lowering effects of certain TP agonists.