Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

2007 ◽  
Vol 293 (1) ◽  
pp. G271-G278 ◽  
Author(s):  
Chitrawina Mahagita ◽  
Steven M. Grassl ◽  
Pawinee Piyachaturawat ◽  
Nazzareno Ballatori
Keyword(s):  
Bile Acid ◽  
Transport Mechanism ◽  
Organic Anion ◽  
Large Family ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Facilitated Diffusion ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporter

Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.

Identification and functional assessment of the novel murine organic anion transporter Oat5 (Slc22a19) expressed in kidney

2004 ◽  
Vol 287 (2) ◽  
pp. F236-F244 ◽  
Author(s):  
Geri L. Youngblood ◽  
Douglas H. Sweet
Keyword(s):  
Ochratoxin A ◽  
Cdna Clone ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Hepatic Expression ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Transporter Family ◽  
2,4 Dichlorophenoxyacetic Acid

An uncharacterized murine cDNA clone was identified and, through sequence, phylogenetic, and functional analysis, determined to encode the newest member of the organic anion transporter family, organic anion transporter 5 (Oat5; Slc22a19). The Oat5 cDNA clone contained an insert 1,964 bp in length with a predicted open reading frame (from bp 84 to bp 1,739) coding for a peptide 551 amino acids long. Slc22a19 was localized to mouse chromosome 19 near the genes encoding Oat1 ( Slc22a6) and Oat3 ( Slc22a8). Northern blot analysis revealed Oat5 is highly expressed in the kidney of adult mice and rats. No sexual dimorphism in renal or hepatic expression of Oat5 was observed. Unlike Oat1–3, Oat5 expression was not detected in the choroid plexus of either mice or rats. Murine Oat5-expressing Xenopus laevis oocytes supported increased accumulation of the mycotoxin ochratoxin A, compared with water-injected control oocytes. This uptake was significantly inhibited by probenecid and the organic anions 2,4-dichlorophenoxyacetic acid, salicylate, and estrone sulfate but not by para-aminohippurate or urate. Transport of ochratoxin A by murine Oat5 was saturable, with an estimated Km of 2.0 ± 0.45 μM. Oat5-mediated transport was neither cis-inhibited nor trans-stimulated by the dicarboxylate glutarate. Uptake was also completely unaffected by short-circuiting of the membrane potential. Thus the motive forces behind Oat5 function, which provide insight into its membrane localization, need to be further resolved. These data demonstrate for the first time that this newly identified gene encodes a protein that functions as an organic anion transporter.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+gradient

2003 ◽  
Vol 284 (4) ◽  
pp. F763-F769 ◽  
Author(s):  
Douglas H. Sweet ◽  
Lauretta M. S. Chan ◽  
Ramsey Walden ◽  
Xiao-Ping Yang ◽  
David S. Miller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Organic Anion ◽  
Renal Proximal Tubule ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Renal Proximal Tubule Cells ◽  
Rat Renal Cortical Slices ◽  
Cortical Slices

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na+ gradient through Na+-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na+-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li+ or methylsuccinate (MS) or when Na+ was absent. All these treatments inhibit the Na+-dicarboxylate cotransporter, but not rat Oat3. Li+, MS, and Na+ removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na+ sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li+, MS, taurocholate, and removal of Na+. Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na+ gradient.

scholarly journals Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1

2016 ◽  
Vol 310 (9) ◽  
pp. F821-F831 ◽  
Author(s):  
Da Xu ◽  
Haoxun Wang ◽  
Qiang Zhang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Organic Anion ◽  
The Body ◽  
Organic Anion Transporter ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter ◽  
In Cells

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

scholarly journals A role for the organic anion transporter OAT3 in renal creatinine secretion in mice

2012 ◽  
Vol 302 (10) ◽  
pp. F1293-F1299 ◽  
Author(s):  
Volker Vallon ◽  
Satish A. Eraly ◽  
Satish Ramachandra Rao ◽  
Maria Gerasimova ◽  
Michael Rose ◽  
...  
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Tubular Secretion ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Mean Values ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Neutral Compounds ◽  
Quantitative Contribution

Tubular secretion of the organic cation, creatinine, limits its value as a marker of glomerular filtration rate (GFR) but the molecular determinants of this pathway are unclear. The organic anion transporters, OAT1 and OAT3, are expressed on the basolateral membrane of the proximal tubule and transport organic anions but also neutral compounds and cations. Here, we demonstrate specific uptake of creatinine into mouse mOat1- and mOat3-microinjected Xenopus laevis oocytes at a concentration of 10 μM (i.e., similar to physiological plasma levels), which was inhibited by both probenecid and cimetidine, prototypical competitive inhibitors of organic anion and cation transporters, respectively. Renal creatinine clearance was consistently greater than inulin clearance (as a measure of GFR) in wild-type (WT) mice but not in mice lacking OAT1 ( Oat1−/−) and OAT3 ( Oat3−/−). WT mice presented renal creatinine net secretion (0.23 ± 0.03 μg/min) which represented 45 ± 6% of total renal creatinine excretion. Mean values for renal creatinine net secretion and renal creatinine secretion fraction were not different from zero in Oat1−/− (−0.03 ± 0.10 μg/min; −3 ± 18%) and Oat3−/− (0.01 ± 0.06 μg/min; −6 ± 19%), with greater variability in Oat1−/−. Expression of OAT3 protein in the renal membranes of Oat1−/− mice was reduced to ∼6% of WT levels, and that of OAT1 in Oat3−/− mice to ∼60%, possibly as a consequence of the genes for Oat1 and Oat3 having adjacent chromosomal locations. Plasma creatinine concentrations of Oat3−/− were elevated in clearance studies under anesthesia but not following brief isoflurane anesthesia, indicating that the former condition enhanced the quantitative contribution of OAT3 for renal creatinine secretion. The results are consistent with a contribution of OAT3 and possibly OAT1 to renal creatinine secretion in mice.

scholarly journals Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytesThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

2010 ◽  
Vol 88 (6) ◽  
pp. 682-691 ◽  
Author(s):  
J. Craig Hartman ◽  
Kenneth Brouwer ◽  
Arun Mandagere ◽  
Lawrence Melvin ◽  
Richard Gorczynski
Keyword(s):  
Organic Anion ◽  
Hepatic Uptake ◽  
Organic Anion Transporter ◽  
Endothelin Receptor Antagonists ◽  
Endothelin Receptor ◽  
Transport Activity ◽  
Receptor Antagonists ◽  
P Glycoprotein ◽  
Anion Transporter ◽  
Hepatic Transporters

To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [3H]estradiol-17β-d-glucuronide (for OATP), and [2-d-penicillamine, 5-d-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 µmol·L–1 for inhibition and at 2 µmol·L–1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%–100% of control), whereas bosentan was most affected (32%–58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

scholarly journals OATP1B3-1B7, a novel organic anion transporting polypeptide, is modulated by FXR ligands and transports bile acids

2019 ◽  
Vol 317 (6) ◽  
pp. G751-G762 ◽  
Author(s):  
Vanessa Malagnino ◽  
Janine Hussner ◽  
Ali Issa ◽  
Angela Midzic ◽  
Henriette E. Meyer zu Schwabedissen
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acid ◽  
Bile Acids ◽  
Smooth Endoplasmic Reticulum ◽  
Organic Anion ◽  
Farnesoid X Receptor ◽  
Organic Anion Transporter ◽  
Organic Anion Transporting Polypeptide ◽  
Anion Transporter ◽  
Solute Carrier

Organic anion transporting polypeptide (OATP) 1B3–1B7 (LST-3TM12) is a member of the OATP1B [solute carrier organic anion transporter ( SLCO) 1B] family. This transporter is not only functional but also expressed in the membrane of the smooth endoplasmic reticulum of hepatocytes and enterocytes. OATP1B3–1B7 is a splice variant of SLCO1B3 in which the initial part is encoded by SLCO1B3, whereas the rest of the mRNA originates from the gene locus of SLCO1B7. In this study, we not only showed that SLCO1B3 and the mRNA encoding for OATP1B3–1B7 share the 5′ untranslated region but also that silencing of an initial SLCO1B3 exon lowered the amount of SLCO1B3 and of SLCO1B7 mRNA in Huh-7 cells. To validate the assumption that both transcripts are regulated by the same promoter we tested the influence of the bile acid sensor farnesoid X receptor (FXR) on their transcription. Treatment of Huh-7 and HepaRG cells with activators of this known regulator of OATP1B3 not only increased SLCO1B3 but also OATP1B3–1B7 mRNA transcription. Applying a heterologous expression system, we showed that several bile acids interact with OATP1B3–1B7 and that taurocholic acid and lithocholic acid are OATP1B3–1B7 substrates. As OATP1B3–1B7 is located in the smooth endoplasmic reticulum, it may grant access to metabolizing enzymes. In accordance are our findings showing that the OATP1B3–1B7 inhibitor bromsulphthalein significantly reduced uptake of bile acids into human liver microsomes. Taken together, we report that OATP1B3–1B7 transcription can be modulated with FXR agonists and antagonists and that OATP1B3–1B7 transports bile acids. NEW & NOTEWORTHY Our study on the transcriptional regulation of the novel organic anion transporting polypeptide (OATP) 1B3–1B7 concludes that the promoter of solute carrier organic anion transporter ( SLCO) 1B3 governs SLCO1B3–1B7 transcription. Moreover, the transcription of OATP1B3–1B7 can be modulated by farnesoid X receptor (FXR) agonists and antagonists. FXR is a major regulator in bile acid homeostasis that links OATP1B3–1B7 to this physiological function. Findings in transport studies with OATP1B3–1B7 suggest that this transporter interacts with the herein tested bile acids.

Transport of cimetidine by flounder and human renal organic anion transporter 1

2003 ◽  
Vol 284 (3) ◽  
pp. F503-F509 ◽  
Author(s):  
Birgitta C. Burckhardt ◽  
Stefan Brai ◽  
Sönke Wallis ◽  
Wolfgang Krick ◽  
Natascha A. Wolff ◽  
...  
Keyword(s):  
Organic Anion ◽  
Human Kidney ◽  
Organic Anion Transporter ◽  
In Vivo Studies ◽  
Xenopus Laevis Oocytes ◽  
Organic Anion Transporters ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Inward Currents

The H2-receptor antagonist cimetidine is efficiently excreted by the kidneys. In vivo studies indicated an interaction of cimetidine not only with transporters for basolateral uptake of organic cations but also with those involved in excretion of organic anions. We therefore tested cimetidine as a possible substrate of the organic anion transporters cloned from winter flounder (fROAT) and from human kidney (hOAT1). Uptake of [3H]cimetidine into fROAT-expressing Xenopus laevis oocytes exceeded uptake into control oocytes. At −60-mV clamp potential, 1 mM cimetidine induced an inward current, which was smaller than that elicited by 0.1 mM PAH. Cimetidine concentrations exceeding 0.1 mM decreased PAH-induced inward currents, indicating interaction with the same transporter. At pH 6.6, no current was seen with 0.1 mM cimetidine, whereas at pH 8.6 a current was readily detectable, suggesting preferential translocation of uncharged cimetidine by fROAT. Oocytes expressing hOAT1 also showed [3H]cimetidine uptake. These data reveal cimetidine as a substrate for fROAT/hOAT1 and suggest that organic anion transporters contribute to cimetidine excretion in proximal tubules.

scholarly journals Dexamethasone induces an imbalanced fetal-placental-maternal bile acid circulation: involvement of placental transporters

BMC Medicine ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Wen Huang ◽  
Jin Zhou ◽  
Juanjuan Guo ◽  
Wen Hu ◽  
Guanghui Chen ◽  
...  
Keyword(s):  
Bile Acid ◽  
Organic Anion ◽  
Maternal Serum ◽  
Farnesoid X Receptor ◽  
Organic Anion Transporter ◽  
Cancer Resistance ◽  
Rat Serum ◽  
Altered Expression ◽  
Anion Transporter ◽  
Fetal Serum

Abstract Background The use of prenatal dexamethasone remains controversial. Our recent studies found that prenatal dexamethasone exposure can induce maternal intrahepatic cholestasis and have a lasting adverse influence on bile acid (BA) metabolism in the offspring. The purpose of this study was to investigate the effects of dexamethasone on fetal-placental-maternal BA circulation during the intrauterine period, as well as its placental mechanism. Methods Clinical data and human placentas were collected and analyzed. Pregnant Wistar rats were injected subcutaneously with dexamethasone (0.2 mg/kg per day) from gestational day 9 to 20. The metabolomic spectra of BAs in maternal and fetal rat serum were determined by LC-MS. Human and rat placentas were collected for histological and gene expression analysis. BeWo human placental cell line was treated with dexamethasone (20–500 nM). Results Human male neonates born after prenatal dexamethasone treatment showed an increased serum BA level while no significant change was observed in females. Moreover, the expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1) and breast cancer resistance protein (BCRP) in the male neonates’ placenta was decreased, while multidrug resistance-associated protein 4 (MRP4) was upregulated. In experimental rats, dexamethasone increased male but decreased female fetal serum total bile acid (TBA) level. LC-MS revealed that primary BAs were the major component that increased in both male and female fetal serum, and all kinds of BAs were significantly increased in maternal serum. The expression of Oatp2b1 and Bcrp were reduced, while Mrp4 expression was increased in the dexamethasone-treated rat placentas. Moreover, dexamethasone increased glucocorticoid receptor (GR) expression and decreased farnesoid X receptor (FXR) expression in the rat placenta. In BeWo cells, dexamethasone induced GR translocation into the nucleus; decreased FXR, OATP2B1, and BCRP expression; and increased MRP4 expression. Furthermore, GR was verified to mediate the downregulation of OATP2B1, while FXR mediated dexamethasone-altered expression of BCRP and MRP4. Conclusions By affecting placental BA transporters, dexamethasone induces an imbalanced fetal-placental-maternal BA circulation, as showed by the increase of primary BA levels in the fetal serum. This study provides an important experimental and theoretical basis for elucidating the mechanism of dexamethasone-induced alteration of maternal and fetal BA metabolism and for exploring early prevention and treatment strategies.

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