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D. F. Woodward, Y. Liang, L. A. Wheeler, M. E. Garst, D. F. Scott, G. Sachs, C. L. Cornell, J. L. Martos, S. N. Pettit, H. G. Fliri; Co-Expression of Prostaglandin FP Receptor and FP Receptor mRNA Splicing Variants Confers Prostamide-Like Pharmacology. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2610.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate prostanoid FP receptor alternative mRNA splicing variants as the putative prostamide receptor
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).
Six new alternative splicing variants of FP receptor mRNA were found in both human and monkey ciliary bodies. Each lacked the 7th 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 PGF2α effects. Finally, AGN 211335 blocked the ocular hypotensive response to bimatoprost but not latanoprost.
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.
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