Carrier-mediated prostaglandin transport has been postulated to occur in many tissues. On the basis of sequence homology, the protein of unknown function encoded by the rat matrin F/G complementary DNA was predicted to be an organic anion transporter. Expression of the matrin F/G complementary DNA in HeLa cells or Xenopus oocytes conferred the property of specific transport of prostaglandins. The tissue distribution of matrin F/G messenger RNA and the sensitivity of matrin F/G-induced prostaglandin transport to inhibitors were similar to those of endogenous prostaglandin transport. The protein encoded by the matrin F/G complementary DNA is thus preferably called PGT because it is likely to function as a prostaglandin transporter.
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.
Newly synthesized prostaglandins (PGs) efflux from cells by simple diffusion, driven by pH and the membrane potential. Metabolic clearance requires energy-dependent uptake across the plasma membrane, followed by cytoplasmic oxidation. Several PG carriers have been cloned and characterized. PGT is broadly expressed in cyclooxygenase (COX)-positive cells, appears to be a lactate/PG exchanger, and is coordinately regulated with COX. By analogy with neurotransmitter release and re-uptake, PGT may regulate pericellular PG levels via re-uptake. PGT may also direct PGs towards and/or away from specific sets of PG receptors. Other members of the OATP transporter family also catalyze PG uptake; these are variably expressed and have variable affinities for PGs. The OATs are alpha-ketoglutarate/organic anion exchangers that accept PGs; these probably represent the uptake step in renal and hepatic PG degradation and excretion. Finally, certain glutathione-conjugated leukotrienes and PGs are actively extruded from cells by the MRPs; these may also play a role in metabolic clearance of PGs.
A Na(+)-independent organic anion transport protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this protein is sufficient for cells to transport the organic anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane protein. This 80-kDa protein migrated as a 65-kDa protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the transporter migrated as an 83-kDa protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this transporter in liver and kidney may have important functional and regulatory consequences.
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