We recently identified a novel prostaglandin transporter called PGT (Kanai, N., Lu, R., Satriano, J. A., Bao, Y., Wolkoff, A. W., and Schuster, V. L. (1995) Science 268, 866 -869). Based 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␣ Ϸ PGE 2 > TXB 2 > > 6 keto-PGF 1␣ . 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 intraoocyte 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). Prostaglandins (PGs)1 and thromboxanes have broad physiologic and pathophysiologic effects, regulating cellular processes in nearly every tissue. They elicit potent actions on the cardiovascular, gastrointestinal, respiratory and reproductive systems, and are important mediators of inflammation, fever, and pain (2). As autacoids, PGs are synthesized by intracellular enzymes at or near their sites of action before they are presented to adjacent PG receptors. Thereafter, extracellular PGs are metabolized in situ within seconds before they are able to reach the general circulation (3, 4). At least in the case of PGE 2 and PGF 2␣ , loss of biologic activity is due to cellular uptake followed by intracellular oxidation (5-8).At physiologic pH, PGs predominate as the charged organic anion (9) and diffuse poorly through the lipid bilayer (10, 11). Facilitated, carrier-mediated PG transport has been demonstrated by many diverse tissues including the lung (8, 12), liver (13), kidney (14), vagina and uterus (15), and blood-brain and blood-intraocular fluid barriers (16).The clearance and metabolism of PGs from the pulmonary circulation has been widely studied using the isolated, perfused rat lung model where concentrative uptake of PGs has been described followed by the appearance of metabolites in the venous effluent (8,17). Substances that inhibit PG transport reduce PG inactivation by the lung (18). Moreover, whereas PGE 1 , PGF 2␣ , PGD 2 , and PGI 2 are all good substrates for the oxidizing enzyme 15-hydroxyprostaglandin dehydrogenase, PGI 2 escapes pulmonary metabolism (8). These phenomena are best explained by selective, carrier-mediated PG tr...

Termination of prostaglandin (PG) signaling has been proposed to involve carrier-mediated uptake across the plasma membrane followed by cytoplasmic oxidation. Here, we tested this hypothesis directly by coexpressing the PG uptake carrier prostaglandin transporter (PGT) in various cell types with and without human PG 15 dehydrogenase (PG15DH). In HeLa cells, which express neither PGT nor PG15DH, exogenously added PGE2 or PGF2␣ were rapidly oxidized to the 13,14-dihydro,15-keto metabolites only when PGT and PG15DH were coexpressed, directly confirming the two-step hypothesis. Cells expressing PG15DH that were broken open formed more PG metabolites than cells in which the PGs could gain access to PG15DH only via PGT. Similar results were obtained using the human prostate cancer cell line LNCaP, in which endogenous PG15DH is induced after exposure to dihydrotestosterone. Because PGT in vivo is expressed in renal collecting duct epithelia, we also expressed PGT in Madin-Darby canine kidney cells grown on filters, where it mediated both the active uptake of PGE2 across the apical membrane and the transepithelial transport of PGE2 to the basolateral compartment. When PG15DH was coexpressed with PGT in these epithelial monolayers, about half of the PGE2 taken up apically was oxidized to 13,14-dihydro,15-keto-PGE2, which in turn exited the cells nondirectionally into both the apical and basolateral compartments. Our data represent reconstitution of the longstanding model of PG metabolism consisting of sequential carrier-mediated PG uptake, cytoplasmic oxidation, and diffusional efflux of the PG metabolite.

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is a broadly expressed transporter of prostaglandins (PGs) and thromboxane that is energetically poised to take up prostanoids across the plasma membrane. To gain insight into the function of PGT, we generated mouse monoclonal antibody 20 against a portion of putative extracellular loop 5 of rat PGT. Immunoblots of endogenous PGT in rat kidney revealed a 65-kDa protein in a zonal pattern corresponding to PG synthesis rates (papilla Х medulla Ͼ cortex). Immunocytochemically, PGT in rat kidneys was expressed in glomerular endothelial and mesangial cells, arteriolar endothelial and muscularis cells, principal cells of the collecting duct, medullary interstitial cells, medullary vasa rectae endothelia, and papillary surface epithelium. Proximal tubules, which are known to take up and metabolize PGs, were negative. Immunoblotting and immunocytochemistry revealed that rat platelets also express abundant PGT. Coexpression of the PG synthesis apparatus (cyclooxygenase) and PGT by the same cell suggests that prostanoids may undergo release and reuptake. carrier proteins; biological transport; molecular cloning PROSTAGLANDINS (PGS) AND THROMBOXANES (Txs) play fundamental roles in context-dependent autocrine and paracrine signaling. In the kidney, for example, depending on the site of release and the receptors activated, PGE 2 vasodilates or vasoconstricts blood vessels, stimulates renin release, and modulates Na, Cl, and water transport (24).The kidney exemplifies the principle that prostanoid synthesis and degradation are compartmentalized into separate cell types and tissue zones. Regionally, the highest rates of renal PG synthesis occur in the papilla. Renal cell types that synthesize PGs and/or express cyclooxygenases (COXs) include glomerular mesangial cells and endothelia, collecting ducts, and medullary interstitial cells (24). In contrast, renal oxidation of PGs occurs in the cortex and juxtamedullary regions (24, 32), primarily by means of the proximal straight tubule, which actively secretes both native and oxidized PGs (13, 16).Our laboratory recently identified a rat cDNA encoding PGT, the first known PG transporter. When expressed heterologously in cultured cells or Xenopus laevis oocytes, PGT mediates the uptake of PGE 2 and TxB 2 , among other eicosanoids (17, 21). The broad expression pattern of PGT mRNA in rats, humans, and mice (17, 21, 28) has suggested a possible physiological role in the release of newly synthesized prostanoids and/or PG uptake before intracellular oxidation (32).To further explore the physiological role of this transporter, we have immunolocalized PGT in rat kidneys and have also sought evidence for PGT expression in rat platelets, which synthesize and release TxA 2 (1). MATERIALS AND METHODS Generation of monoclonal antibody 20 against rat PGT.We PCR-amplified a portion of the rat PGT (rPGT) cDNA corresponding to deduced amino acids 430-505 on putative exofacial loop 5 and cloned it into the vector pGEX to generate an rPGT-glutathione S-transferase (GST) fusion protein. Mice were im...

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