Mechanism of Prostaglandin E2 Transport across the Plasma Membrane of HeLa Cells and Xenopus Oocytes Expressing the Prostaglandin Transporter “PGT”

Abstract

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...

The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs

“…Other observations support this picture: In monolayers of cultured canine cortical collecting tubule cells, for example, PGE 2 crossed the monolayer slowly, at the same rate as the inulin used to measure imperfections in the monolayer (41). Similarly, human cell lines (32,42) and MDCKII cells (43) were impermeable to prostanoids, with no accumulation (or flux) of prostaglandins or thromboxane unless PGT was heterologously expressed. The permeability of Xenopus laevis oocytes, a model system used in many studies of transport proteins, is also low for prostaglandins (32,36,42).…”

“…Similarly, human cell lines (32,42) and MDCKII cells (43) were impermeable to prostanoids, with no accumulation (or flux) of prostaglandins or thromboxane unless PGT was heterologously expressed. The permeability of Xenopus laevis oocytes, a model system used in many studies of transport proteins, is also low for prostaglandins (32,36,42). Despite this low intrinsic permeability, the amount of label released from oocytes injected with [ 3 H]PGE 2 increased linearly with concentration, up to an intraoocyte concentration of 380 nM, suggestive of passive diffusion (42).…”

“…Indeed, the very fact that PGT can be used to accumulate PGE 1 and PGE 2 50-fold in cells (ref. 42 and Fig. 4) shows that diffusion of PGE 1 and PGE 2 out of cells is relatively slow and is no match for an active uptake transporter.…”

Prostaglandin Signaling in the Renal Collecting Duct

“…Similarly, the renal collecting duct expresses apical EP 4 receptors and basolateral EP 1 receptors that signal opposite effects on Na ϩ and water transport (4,5). Since PGE2 likely exits collecting duct cells non-directionally by simple diffusion (37,38), one method for directing PGE2 toward basolateral (EP 1 ) receptors would be to use an apical reuptake carrier like PGT. The reclaimed PGE2 could either be oxidized or could rediffuse intact out of the cell across the basolateral membrane.…”

Prostaglandin transporter expression in mouse brain during development and in response to hypoxia

“…Extracellular PGs exert their major actions via multiple cell surface G-protein coupled receptors (Coleman et al, 1994;Narumiya et al, 1999) and are immediately taken up intracellularly for their inactivation or action via specific nuclear receptors (Zhu et al, 2006). Prostanglandin transport takes place via a prostaglandin transporter (PGT) and this transport plays an important role since, at physiologic pH, PGs exist as charged anions and diffuse poorly across the plasma membrane (Schuster, 1998;Chan et al, 1998). PGT has been identified in various tissues (endothelial cells, liver, kidney, lung, smooth muscle) (Lu et al, 1996) and, in contrast to multiple PG specific receptors and their isoforms (Narumiya et al, 1999), PGT has been shown to have affinity with most major PGs (PGE 2 =PGF 2 >PGD 2 ) (Schuster, 1998).…”