Characterization and identification of steroid sulfate transporters of human placenta

2003 ◽  
Vol 284 (2) ◽  
pp. E390-E398 ◽  
Author(s):  
Bernhard Ugele ◽  
Marie V. St-Pierre ◽  
Monika Pihusch ◽  
Andrew Bahn ◽  
Peer Hantschmann
Keyword(s):  
Organic Anion ◽  
Diffusion Constant ◽  
Physiological Role ◽  
Placental Tissue ◽  
Organic Anion Transporter ◽  
Uptake Mechanism ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
S Uptake ◽  
Steroid Sulfate

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16α-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-35S uptake by MT revealed a saturable uptake mechanism ( K m = 26 μM, V max = 428 pmol · mg protein−1 · min−1), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 μl · mg protein−1 · min−1). Uptake of [3H]DHEA-S by MT was Na+dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [35S]BSP, [3H]estrone-sulfate, but not 3H-labeled ouabain, estradiol-17β-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na+-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.

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.

Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter

2005 ◽  
Vol 97 (4) ◽  
pp. 369-375 ◽  
Author(s):  
Jin-Oh Kwak ◽  
Hyun-Woo Kim ◽  
Kwang-Jin Oh ◽  
Chang-Bo Ko ◽  
Hwayong Park ◽  
...  
Keyword(s):  
Organic Anion ◽  
Organic Anion Transporter ◽  
Sulfate Transporter ◽  
Anion Transporter ◽  
Steroid Sulfate

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 Organic Anion Transporter 1B1: An Important Factor in Hepatic Thyroid Hormone and Estrogen Transport and Metabolism

Endocrinology ◽  
2008 ◽  
Vol 149 (9) ◽  
pp. 4695-4701 ◽  
Author(s):  
Wendy M. van der Deure ◽  
Edith C. H. Friesema ◽  
Frank Jan de Jong ◽  
Yolanda B. de Rijke ◽  
Frank H. de Jong ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Hepatic Transport ◽  
Transfected Cells ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Metabolism Of Bilirubin

Sulfation is an important pathway in the metabolism of thyroid hormone and estrogens. Sulfation of estrogens is reversible by estrogen sulfatase, but sulfation of thyroid hormone accelerates its degradation by the type 1 deiodinase in liver. Organic anion transporters (OATPs) are capable of transporting iodothyronine sulfates such as T4 sulfate (T4S), T3S, and rT3S or estrogen sulfates like estrone sulfate (E1S), but the major hepatic transporter for these conjugates has not been identified. A possible candidate is OATP1B1 because model substrates for this transporter include the bilirubin mimic bromosulfophthalein (BSP) and E1S, and it is highly and specifically expressed in liver. Therefore, OATP1B1-transfected COS1 cells were studied by analysis of BSP, E1S, and iodothyronine sulfate uptake and metabolism. Two Caucasian populations (155 blood donors and 1012 participants of the Rotterdam Scan Study) were genotyped for the OATP1B1-Val174Ala polymorphism and associated with bilirubin, E1S, and T4S levels. OATP1B1-transfected cells strongly induced uptake of BSP, E1S, T4S, T3S, and rT3S compared with mock-transfected cells. Metabolism of iodothyronine sulfates by cotransfected type 1 deiodinase was greatly augmented in the presence of OATP1B1. OATP1B1-Val174 showed a 40% higher induction of transport and metabolism of these substrates than OATP1B1-Ala174. Carriers of the OATP1B1-Ala174 allele had higher serum bilirubin, E1S, and T4S levels. In conclusion, OATP1B1 is an important factor in hepatic transport and metabolism of bilirubin, E1S, and iodothyronine sulfates. OATP1B1-Ala174 displays decreased transport activity and thereby gives rise to higher bilirubin, E1S, and T4S levels in carriers of this polymorphism.

Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules

2004 ◽  
Vol 287 (5) ◽  
pp. F1021-F1029 ◽  
Author(s):  
S. Soodvilai ◽  
V. Chatsudthipong ◽  
K. K. Evans ◽  
S. H. Wright ◽  
W. H. Dantzler
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Anion Transport ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Dependent Manner ◽  
Organic Anion Transport ◽  
Renal Proximal Tubules ◽  
Estrone Sulfate ◽  
Anion Transporter

We investigated the regulation of organic anion transport driven by the organic anion transporter 3 (OAT3), a multispecific OAT localized at the basolateral membrane of the renal proximal tubule. PMA, a PKC activator, inhibited uptake of estrone sulfate (ES), a prototypic substrate for OAT3, in a dose- and time-dependent manner. This inhibition was reduced by 100 nM bisindoylmaleimide I (BIM), a specific PKC inhibitor. The α1-adrenergic receptor agonist phenylephrine also inhibited ES uptake, and this effect was reduced by BIM. These results suggest that PKC activation downregulates OAT3-mediated organic anion transport. In contrast, epidermal growth factor (EGF) increased ES uptake following activation of MAPK. Exposure to PGE2 or dibutyryl (db)-cAMP also enhanced ES uptake. Stimulation produced by PGE2 and db-cAMP was prevented by the PKA inhibitor H-89, indicating that this stimulation required PKA activation. In addition, inhibition of cyclooxygenase 1 (COX1) (but not COX2) inhibited ES uptake. Furthermore, the stimulatory effect of EGF was eliminated by inhibition of either COX1 or PKA. These data suggest that EGF stimulates ES uptake by a process in which MAPK activation results in increased PGE2 production that, in turn, activates PKA and subsequently stimulates ES uptake. Interestingly, EGF did not induce upregulation immediately following phenylephrine-induced downregulation; and phenylephrine did not induce downregulation immediately after EGF-induced upregulation. These data are the first to show the regulatory response of organic anion transport driven by OAT3 in intact renal proximal tubules.

Organic anion transport in choroid plexus from wild-type and organic anion transporter 3 (Slc22a8)-null mice

2004 ◽  
Vol 286 (5) ◽  
pp. F972-F978 ◽  
Author(s):  
Destiny Sykes ◽  
Douglas H. Sweet ◽  
Simon Lowes ◽  
Sanjay K. Nigam ◽  
John B. Pritchard ◽  
...  
Keyword(s):  
Choroid Plexus ◽  
Organic Anion ◽  
Anion Transport ◽  
Organic Anion Transporter ◽  
Null Mouse ◽  
Organic Anion Transport ◽  
Wild Type ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Organic Anion Transporter 3

The choroid plexus actively transports endogenous, xenobiotic, and therapeutic compounds from cerebrospinal fluid to blood, thereby limiting their exposure to the central nervous system (CNS). Establishing the mechanisms responsible for this transport is critical to our understanding of basic choroid plexus physiology and will likely impact drug targeting to the CNS. We recently generated an organic anion transporter 3- (Oat3)-null mouse, which exhibited loss of PAH, estrone sulfate, and taurocholate transport in kidney and of fluorescein (FL) transport in choroid plexus. Here, we measured the uptake of four Oat3 substrates by choroid plexus from wild-type and Oat3-null mice to establish 1) the contribution of Oat3 to the apical uptake of each substrate and 2) the Na dependence of transport by Oat3 in the intact tissue. Mediated transport of PAH and FL was essentially abolished in tissue from Oat3-null mice. In contrast, only a 33% reduction in estrone sulfate uptake was observed in tissue from Oat3-null mice and, surprisingly, no reduction in taurocholate uptake could be detected. For PAH, FL, and estrone sulfate, all Oat3-mediated transport was Na dependent. However, estrone sulfate and taurocholate also exhibited additional mediated and Na-dependent components of uptake that were not attributed to Oat3, demonstrating the complexity of organic anion transport in this tissue and the need for further examination of expressed transporters and their energetics.

Impact of Organic Anion Transporter 3 (OAT3) on Bendamustine Uptake of Lymphoma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4184-4184
Author(s):  
Yohannes Hagos ◽  
Gerald G. Wulf ◽  
Vladimir Shnitsar ◽  
Philip Hundertmark ◽  
Shvangi Gupta ◽  
...  
Keyword(s):  
Cell Lines ◽  
Organic Anion ◽  
Inhibitory Effect ◽  
Lymphoma Cell ◽  
Organic Anion Transporter ◽  
Hek293 Cells ◽  
Sulfate Uptake ◽  
Lymphoma Cells ◽  
Estrone Sulfate ◽  
Anion Transporter

Abstract One main problem of tumor therapy is the resistance of malignant cells to cytostatics due to high expression of efflux transporters. Whereas the role of these efflux transporters for tumor cell resistance is well established, little is known about uptake transporters, which may increase the sensibility of tumor cells for cytostatics. In the present study we addressed the interaction of cytostatics established for the treatment of lymphoma, namely melphalan, chlorambucil or bendamustine with human Organic Anion Transporter (OATs), which belong to the Solute carrier (SLC) gene family. We selected these cytostatics, because they show structural similarity to p-aminohyppurate (PAH), the model substrate of OATs. OATs are mainly expressed in the kidney, where they are responsible for the excretion of endogenous and exogenous organic anions like urate or various drugs e.g. diuretics. Initially, we examined the cis-inhibitory effect of melphalan, chlorambucil and bendamustine on OAT1-mediated [3H]PAH uptake as well as OAT3- and OAT4- mediated [3H]estrone sulfate uptake in HEK293 cells, which were stably transfected with these transporters. Melphalan did not show any significant inhibitory effect on all tested OATs. 100 μM chlorambucil reduced OAT1-, OAT3- and OAT4-mediated uptake of PAH or estrone sulfate down to 14.6 ± 0.17%, 16.3 ± 4.0% and 66.0 ± 1.4%, respectively. 100 μM bendamustine inhibited only OAT3-mediated estrone sulfate uptake up to 91.9 ± 0.5% compared to control cells. OAT1- or OAT4- facilitated transport of PAH and estrone sulfate remained unchanged by bendamustine, suggesting that bendamustine interacts exclusively with OAT3. To determine the affinity of OAT3 for bendamustine and chlorambucil, we performed concentration dependent inhibition of OAT3-mediated estrone sulfate uptake and calculated the Ki values for both cytostatics. Dixon-Plot evaluation confirmed a competitive inhibition of OAT3 by bendamustine as well as chlorambucil. The results demonstrated higher affinity of OAT3 for bendamustine with a Ki value of 2.7 μM than for chlorambucil, showing a Ki value of 38.2 μM. To elucidate the expression of OATs in lymphoma cell lines, we performed RT-PCR experiments. Our data demonstrate high expression of OAT3 in all cell lines compared to lymphocytes isolated from a normal person. No expression of OAT1 and OAT4 was observed any lymphoma cell lines. The expression of OAT3 in B-cell lymphoma cell lines Karpas, Raji, SudHL4 and T-cell lymphoma cell lines L428, Jurkat and Hut78 was quantified by real time PCR. The highest expression of OAT3 was observed in the order Jurkat>Hut78>SudHL4>L428>Raji>Karpas. The expression of OAT3 was confirmed by real time PCR in four patients with chronic lymphocytic leukamia. OAT3- dependent cytostatic effects of bendamustine was examined by [3H] thymidine incorporation. 30 min incubation of OAT3-expressing HEK293 cells with 10, 50 or 100 μM bendamustine significantly reduced the proliferation of transfected versus non-transfected cells. We conclude that the molecular background for the cytostatic efficiency of bendamustine in lymphoma cells is due to 1) the expression of OAT3 in lymphoma cells and 2) a the high affinity of OAT3 for bendamustine.

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