Co-Expression of Prostaglandin FP Receptor and FP Receptor mRNA Splicing Variants Confers Prostamide-Like Pharmacology

Abstract

Purpose: : To investigate prostanoid FP receptor alternative mRNA splicing variants as the putative prostamide receptor

Methods: : RT-PCR analyses were performed on human and monkey ciliary body cDNA libraries using FP receptor specific primers. Novel PCR products were subcloned and sequenced. Stable transfectants of wt. FP receptors, FP receptor mRNA alternative splicing variants (alt.FP) and co-transfections of FP/alt-FP were prepared in HEK 293/EBNA cells using retrovirus vector. Northern blots, MLC phophorylation, and Ca²⁺ signaling (FLIPR) experiments adhered to standard protocols. Isolated tissue pharmacology was performed using feline iris sphincter smooth muscle. Pneumatonometry was conducted in conscious dogs (BASi, Evansville, USA).

Results: : Six new alternative splicing variants of FP receptor mRNA were found in both human and monkey ciliary bodies. Each lacked the 7^th TM spanning segment, the carboxyl termini were extracellular, and they appeared functionally inert. When co-expressed with wt FP receptors, prostamide-like activity was conferred. Bimatoprost exhibited no meaningful activity in FP receptor transfects but in FP/alt FP co-transfects it produced a pronounced Ca²⁺ transient followed by a second Ca²⁺ wave. The secondary Ca²⁺ response was susceptible to the prostamide antagonist AGN 211335 (pA₂ = 7.5 in feline iris). Similarly, Cyr 61 upregulation and MLC phosphorylation produced by bimatoprost in FP/alt FP co-transfectants was susceptible to AGN 211335. AGN 211335 did not alter PGF_2α effects. Finally, AGN 211335 blocked the ocular hypotensive response to bimatoprost but not latanoprost.

Conclusions: : These data suggest that the effects of prostamides, as determined by their susceptibility to AGN 211334-6, may be mediated by a wt FP/alt FP interaction. Preliminary evidence suggests that FP/alt FP may form a heterodimeric association.

Keywords: receptors: pharmacology/physiology • eicosanoids • intraocular pressure