Conserved from fish to mammals, renal proximal tubule organic anion secretion plays an important role in drug and xenobiotic elimination. Studies with the model substrate p-aminohippurate (PAH) have suggested that a basolateral PAH/α-ketoglutarate exchanger imports diverse organic substrates into the proximal tubule prior to apical secretion. cDNAs encoding PAH transporters have been cloned recently from rat and flounder. Here we report the cloning of a highly similar human PAH transporter (hPAHT) from human kidney. By Northern blot analysis and EST database searching, hPAHT mRNA was detected in kidney and brain. PCR-based monochromosomal somatic cell hybrid mapping placed the hPAHT gene on chromosome 11. When expressed transiently in vitro, hPAHT catalyzed time-dependent and saturable [3H]PAH uptake ( K m of ∼5 μM). Preincubation with unlabeled α-ketoglutaric or with glutaric acid stimulated tracer PAH uptake, and preincubation with unlabeled PAH stimulated tracer α-ketoglutarate uptake, results that are consistent with PAH/α-ketoglutarate exchange. Several structurally diverse organic anions cis-inhibited PAH uptake. Like rat OAT1 organic anion transporter, hPAHT was inhibited by furosemide, indomethacin, probenecid, and α-ketoglutarate. Unlike OAT1, hPAHT was not inhibited by prostaglandins or methotrexate (MTX). Moreover, tracer PGE2 and MTX were not transported, indicating that the substrate specificity for transport by hPAHT is not broad. PAH uptake was inhibited by phorbol 12-myristate 13-acetate (PMA) in a dose- and time-dependent fashion, but not by the inactive 4α-phorbol-12,13 didecanoate. PMA-induced inhibition was blocked by staurosporine. Thus the protein kinase C-mediated inhibition of basolateral organic anion entry previously reported in intact tubules is likely due, at least in part, to direct modulation of the PAH/α-ketoglutarate exchanger.

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...

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...

We previously characterized the prostaglandin (PG) transporter PGT as an exchanger in which [(3)H]PGE(2) influx is coupled to the efflux of a countersubstrate. Here, we cultured HeLa cells that stably expressed human PGT under conditions known to favor glycolysis (glucose as a carbon source) or oxidative phosphorylation (glutamine as a carbon source) and studied the effect on PGT-mediated [(3)H]PGE(2) influx. PGT-expressing cells grown in glutamine exhibited a 2- to 4-fold increase in [(3)H]PGE(2) influx compared with the antisense control, whereas cells grown in glucose exhibited a 14-fold increase. In the presence of 10 vs. 25 mM glucose during the uptake, there was a dose-dependent increment in [(3)H]PGE(2) influx. Cis inhibition of [(3)H]PGE(2) influx occurred with lactate at physiological concentrations (apparent K(m) = 48 +/- 12 mM). Preloading with lactate caused a dose-dependent trans stimulation of PGT-mediated [(3)H]PGE(2) uptake, and external lactate caused trans stimulation of PGT-mediated [(3)H]PGE(2) release. Together, these data are consistent with PGT-mediated PG-lactate exchange. Cells engaged in glycolysis would then be poised energetically for prostanoid uptake by means of PGT.

We recently identified and/or cloned the PG transporter PGT in the rat (rPGT) (Kanai, N., R. Lu, J. A. Satriano, Y. Bao, A. W. Wolkoff, and V. L. Schuster, Science 268: 866–869, 1995) and the human (hPGT) (Lu, R., and V. L. Schuster, J. Clin. Invest. 98: 1142–1149, 1996). Here we have cloned and expressed the mouse PGT (mPGT) cDNA. The tissue distribution of mPGT mRNA expression is significantly more restricted than that of rPGT and hPGT mRNA. Although the deduced amino acid sequence of mPGT is similar to the rat (91% identity) and human (82% identity) homologues, it has three regions of dissimilarity: amino acids 128–163 and 283–298, and valine 610 and isoleucine 611 (predicted to lie within putative transmembrane span 12). Affinities of hPGT, rPGT, and mPGT for several PG substrates differed, with hPGT having the highest [low Michaelis constant ( K m)] and mPGT the lowest affinity. A chimeric protein, linking the N-terminal domain of mPGT with the C-terminal domain of hPGT, had affinity for PGE2 indistinguishable from that of hPGT, indicating that the C-terminal domain dictates K m. We mutagenized mouse valine 610 and isoleucine 611 to their corresponding human residues (methionine and glycine, respectively); however, these changes did not convert the inhibition constant of mPGT to that of hPGT. The mouse gene was localized to chromosome 9 in a region syntenic with the region of human chromosome 3 containing the hPGT gene. These studies highlight the species-dependence of tissue expression and function of PGT and lay the groundwork for the use of the mouse as a model system for the study of PGT function.