Mechanism of prostaglandin E2 transport across the plasma membrane of HeLa cells and Xenopus oocytes expressing the prostaglandin transporter "PGT"

We recently identified a novel prostaglandin transporter called PGT (Banal, N., Lu, R., Satriano, J.A., Bao, Y., Wolkoff, A. W, and Schuster, V. L. (1995) Science 268, 866-869). Eased on initial functional studies, we have hypothesized that PGT might mediate the release of newly synthesized prostaglandins (PG), epithelial transport of PGs, or metabolic clearance of PGs. Here we examined the mechanism of PGT transport as expressed in HeLa cells and Xenopus oocytes, using isotopic PG influx and efflux studies. In both native HeLa cells and oocytes, cell membranes were poorly permeable to PGs. In contrast, in oocytes injected with PGT mRNA, the PG influx permeability coefficient was 90-157 times that of oocytes injected with water. The rank order substrate profile was PGF(2 alpha) approximate to PGE(2) > TXB2 much greater than 6 keto-PGF(1 alpha). PG influx displayed an overshoot with rapid accumulation of tracer PGE(2), followed by a gradual return to baseline. Based on estimated oocyte volumes, the PGT-mediated accumulation of PGE(2) reached steady state at intra-oocyte concentrations 25-fold higher than the external media. The accumulation of PG was not due to intracellular binding or metabolism. PGT-mediated uptake was ATP-and temperature-dependent, but not sodium-dependent, and was inhibited by disulfonic stilbenes, niflumic acid, and the thiol reactive anion MTSES (Na(2-sulfonatoethyl)methanethiosulfonate). [H-3]PGE(2) efflux from PGT-transfected HeLa cells was stimulated by external (trans) PGE(2) in a dose-dependent fashion and was inhibited by bromcresol green and 4,4'-diisothiocyana tostilbene-2,2'-disulfonate. Membrane depolarization inhibited uptake of [H-3]PGE(2), consistent with a model of net outward movement of negative charge during the translocation event. These findings suggest that PGT mediates [H-3]PGE(2) accumulation via obligatory, electrogenic anion exchange.