Differences in transport function of the human and rat orthologue of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1).

The human drug transporter Organic Anion Transporting Polypeptide (hOATP)2B1 facilitates cellular uptake of its substrates. Various studies suggest that hOATP2B1 is involved in intestinal absorption, but preclinical evaluations performed in rodents do not support this. Thus, our study aimed to compare the expression and function of hOATP2B1 with its orthologue in rats (rOatp2b1). Even if the general expression pattern was comparable, the transporters exhibited substantial differences on functional level. While bromosulfophthalein and atorvastatin were substrates of both transporters, the steroid sulfate conjugates estrone 3-sulfate (E1S), progesterone sulfate and dehydroepiandrosterone sulfate were only transported by hOATP2B1. To further elucidate these functional differences, experiments searching for the E1S substrate recognition site were conducted generating human-rat chimera as well as partly humanized variants of rOatp2b1. The rOatp2b1-329-hOATP2B1 chimera led to a significant increase in E1S uptake suggesting the C-terminal part of the human transporter is involved. However, humanization of various regions within this part, namely of the transmembrane domain (TMD)-9, TMD-10 or the extracellular loop-5 did not significantly change E1S transport function. Replacement of the intracellular loop-3, slightly enhanced cellular accumulation of sulfated steroids. Taken together, we report that OATP2B1 exhibited differences in recognition of steroid sulfate conjugates comparing the rat and human orthologues.

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

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.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

LST-3TM12 is a member of the OATP1B family and a functional transporter.

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2 pmol mg-1 min-1; Km 34.2 µm) and estradiol 17ß-glucuronide (Vmax 29.9 mol mg-1 min-1 and Km 32.8 µM). In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.