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 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.

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.

scholarly journals Hepatic Uptake of Bilirubin and Its Conjugates by the Human Organic Anion Transporter SLC21A6

2000 ◽  
Vol 276 (13) ◽  
pp. 9626-9630 ◽  
Author(s):  
Yunhai Cui ◽  
Jörg König ◽  
Inka Leier ◽  
Ulrike Buchholz ◽  
Dietrich Keppler
Keyword(s):  
Organic Anion ◽  
Hepatic Uptake ◽  
Organic Anion Transporter ◽  
Anion Transporter

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.

Cisplatin induces renal expression of P-glycoprotein and canalicular multispecific organic anion transporter

1999 ◽  
Vol 277 (6) ◽  
pp. F832-F840 ◽  
Author(s):  
Michel Demeule ◽  
Mathieu Brossard ◽  
Richard Béliveau
Keyword(s):  
Chemotherapeutic Agent ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Rat Tissues ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Anion Transporter ◽  
Renal Brush Border Membranes ◽  
Renal Expression ◽  
Renal Brush Border

The expression of two members of the ATP-binding cassette family of transport proteins, P-glycoprotein (P-gp) and the canalicular multispecific organic anion transporter (cMOAT or Mrp2), was evaluated in renal brush-border membranes (BBM) and various rat tissues after cisplatin treatment. One administration of cisplatin (5 mg/kg) increased P-gp expression by >200–300% in renal BBM and in crude membranes from liver and intestine. The increase in P-gp expression in the kidney was also detected in photolabeling experiments, suggesting the induction of functional P-gp. cMOAT expression was increased by >10-fold in renal BBM after cisplatin administration, although it had no effect on liver cMOAT expression. The increase in the levels of both proteins was maximal at 2 days after cisplatin treatment and lasted for at least 8 days. These results indicate that a single administration of cisplatin induces overexpression of P-gp and cMOAT in specific tissues. This may be of significant relevance to the design of clinical trials using cisplatin as a single chemotherapeutic agent or in combination with other drugs.

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