Heterologous Expression and Functional Characterization of a Mouse Renal Organic Anion Transporter in Mammalian Cells

Abstract

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and posttranslational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, antihypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics

“…1A). This is consistent with previous studies showing that coincubation of mOat1-transfected oocytes with unlabeled PAH resulted in dosedependent inhibition of uptake of labeled PAH with an estimated K i of 13 M (11), which is similar to previously reported values for the affinity of PAH for mOat1 (22). In comparison, ES inhibited tracer uptake with an IC 50 value for mOat3 of 8 Ϯ 2 M (Fig.…”

Decreased Renal Organic Anion Secretion and Plasma Accumulation of Endogenous Organic Anions in OAT1 Knock-out Mice

“…(Expression of mouse OAT1 in oocytes was induced by microinjection of the corresponding in vitro transcribed cRNA.) As expected, co-incubation of oocytes with unlabeled PAH resulted in dosedependent inhibition of uptake of labeled PAH, with an estimated K i of ϳ13 M (this is similar to previously reported values for the affinity of PAH for OAT1 (28,69)). …”

Isolation, Characterization and Differential Gene Expression of Multispecific Organic Anion Transporter 2 in Mice

“…Although several OAT isoforms have been isolated to date, information of the function of each isoform is still not sufficient. Moreover, the functional property of mouse OAT homologs is limited despite the fact that mouse OAT1 (mOAT1) (NKT) and mouse OAT3 (mOAT3) (Roct) isoforms have been isolated (Lopez-Nieto et al, 1997;Brady et al, 1999;Kuze et al, 1999). Among the OAT isoforms, only OAT2 is predominantly expressed in the liver, and this isoform is considered one of the key molecules in hepatic handling of organic anions.…”

“…OAT1 has a broad substrate specificity and interacts with a wide range of organic anions such as p-aminohippuric acid (PAH), dicarboxylates, cyclic nucleotides, prostaglandin E 2 (PGE 2 ) (Sekine et al, 1997;Sweet et al, 1997;Apiwattanakul et al, 1999). Subsequently, OAT1 orthologs have been isolated from humans and mice (LopezNieto et al, 1997;Hosoyamada et al, 1999;Kuze et al, 1999;Lu et al, 1999;Race et al, 1999). So far, four additional members belonging to the organic anion transporter family, OAT2 (Simonson et al, 1994;Sekine et al, 1998), OAT3 (Kusuhara et al, 1999;Race et al, 1999;Cha et al, 2001), OAT4 (Cha et al, 2000), and OAT5 (Sun et al, 2001), have been cloned and identified.…”