Characterization of Prostaglandin EP4 Receptors in Human Conventional Outflow Cells

Abstract

Purpose: : Prostaglandin EP₄ receptors are expressed by both trabecular meshwork (TM) and Schlemm’s canal (SC) cells of the conventional outflow pathway. Activation of EP₄ receptors in monkeys efficaciously lowers intraocular pressure, while having no effect on aqueous secretion or uveoscleral outflow. We hypothesize that prostaglandin EP₄ receptors regulate human conventional outflow.

Methods: : To test this idea, cAMP accumulation was used as an indicator of receptor activation in primary cultures of confluent and mature human TM and SC cells that were treated with increasing concentrations of a selective EP₄ agonist (3,7-dithiaPGE₁) with or without forskolin (FSK). HEK 293 cells stably transfected with prostaglandin EP₄ and EP₂ receptors were used as controls. Intracellular cAMP accumulation was measured using a PKA binding assay and data were analyzed using GraphPad statistical analysis software.

Results: : Treatment with 3,7-dithiaPGE₁ resulted in a dose-related increase in cAMP accumulation in both SC (EC₅₀=9.76 x 10⁶, n=4) and TM (EC₅₀=8.66 x 10⁷, n=6) cells. By comparison, 3,7-dithiaPGE₁ treatment increased cAMP accumulation in both EP₄ HEK 293 cells (EC₅₀=2.85 x 10¹⁰, n=4) and EP₂ HEK293 cells (EC₅₀=3.9 x 10⁷, n=4), exhibiting a 1000 fold difference in relative potency. Interestingly, other studies showed that 3,7-dithiaPGE₁ treatment affects cAMP accumulation in FSK-pretreated SC cells in a dose-dependent manner at concentrations corresponding to prostaglandin EP₄ receptor activity (n=4).

Keywords: trabecular meshwork • receptors: pharmacology/physiology