Regulation of prostaglandin EP₁ and EP₄ receptor signaling by carrier-mediated ligand reuptake

After synthesis and release from cells, prostaglandin E₂ (PGE₂) undergoes reuptake by the prostaglandin transporter (PGT), followed by cytoplasmic oxidation. Although genetic inactivation of PGT in mice and humans results in distinctive phenotypes, and although experiments in localized environments show that manipulating PGT alters downstream cellular events, a direct mechanistic link between PGT activity and PGE₂ (EP) receptor activation has not been made. Toward this end, we created two reconstituted systems to examine the effect of PGT expression on PGE₂ signaling via two of its receptors (EP₁ and EP₄). In human embryonic kidney cells engineered to express the EP₁ receptor, exogenous PGE₂ induced a dose-dependent increase in cytoplasmic Ca²⁺. When PGT was expressed at the plasma membrane, the PGE₂ dose–response curve was right-shifted, consistent with reduction in cell surface PGE₂ availability; a potent PGT inhibitor acutely reversed this shift. When bradykinin was used to induce endogenous PGE₂ release, PGT expression similarly induced a reduction in Ca²⁺ responses. In separate experiments using Madin–Darby Canine Kidney cells engineered to express the PGE₂ receptor EP₄, bradykinin again induced autocrine PGE₂ signaling, as judged by an abrupt increase in intracellular cAMP. As in the EP₁ experiments, expression of PGT at the plasma membrane caused a reduction in bradykinin-induced cAMP accumulation. Pharmacological concentrations of exogenous PGE₂ induced EP₄ receptor desensitization, an effect that was mitigated by PGT. Thus, at an autocrine/paracrine level, plasma membrane PGT regulates PGE₂ signaling by decreasing ligand availability at cell surface receptors.