P341 MOLECULAR IDENTIFICATION OF A NOVEL PROSTAGLANDIN E2 TRANSPORTER

Rika Hayashi , Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
Katsuhisa Inoue , Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
Kinya Ohta , Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
Hiroaki Yuasa , Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

Prostaglandin E2 (PGE2) is a primary product of arachidonic metabolism, and plays a key role in the regulation of physiological and pathological homeostasis within the body. PGE2 is synthesized by the sequential actions of cyclooxygenase-1 and -2, and specific prostaglandin synthases, and then released outside the cells. Since PGE2 is a charged anion and diffuses poorly across the cellular membranes, carrier-mediated transport systems are needed for its efficient trafficking. However, the export mechanism of prostaglandins has not been fully elucidated yet. Therefore, we attempted to identify one of transporters involved in the export of prostaglandins synthesized intracellularly. In the present study, we selected the candidate genes that may encode a prostaglandin transporter by in silico screening and identified the one that is expressed in LNCaP human prostate adenocarcinoma cells, which are capable of producing prostaglandins. Its cDNA cloned from LNCaP cells was introduced into Madin-Darby canine kidney (MDCK) II cells for stable transfection. Functional characterization of the transporter, which is herein named prostaglandin efflux transporter (PGET), was conducted by evaluation of the uptake of [3H]PGE2 into the transfected cells. The specific uptake of PGE2 mediated by PGET was saturable with a Michaelis constant of 24 µM, which indicates a fairly high affinity. The specific uptake was found not to depend on Na+, but was dependent on the extracellular pH, being optimal at acidic pHs. At pH 7.4, interestingly, PGE2 preloaded in the cells was found to be rapidly effluxed. Therefore, PGET may be involved in the export of PGE2 from the cells physiologically. PGET-mediated PGE2 uptake was inhibited by sulfobromophthalein, an inhibitor of anion transporters, indicating its characteristic as an anion-sensitive transporter for PGE2. Furthermore, it was found that many other prostaglandins, except for arachidonate, inhibited PGE2 uptake by PGET, suggesting that PGET can specifically recognize prostaglandin derivatives, and mediate their transports. Thus we have successfully identified PGET as a novel prostaglandin transporter that may be involved in the export of prostaglandins from the cells. PGET may play a role in the cell signalings by prostaglandins by modulating their extracellular concentrations.