Abstract
17α-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-Km (Michaelis constant) kinetics (Km = 21.1 μM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-Km model (Km1 = 1.6 μM; Km2 = 195 μM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.031526.
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ABBREVIATIONS:
- EE2
- 17α-ethinylestradiol
- EE2-Sul
- 17α-ethinylestradiol-3-O-sulfate
- EE2-Glu
- 17α-ethinylestradiol-3-O-glucuronide
- OAT
- organic anion transporter
- NTCP
- sodium-taurocholate cotransporting polypeptide
- BCRP
- breast cancer resistance protein
- SLC
- solute carrier
- ABC
- ATP-binding cassette
- OATP
- organic anion-transporting polypeptide
- OCT
- organic cation transporter
- MRP
- multidrug resistance-associated protein
- MATE1
- multidrug and toxin extrusion
- MDCK
- Madin-Darby canine kidney
- HEK
- human embryonic kidney
- PAH
- para-aminohippurate
- MPP
- 1-methyl-4-phenylpyridinium
- FRT
- Flp recombination target
- PAPS
- 3′-phosphoadenosine 5′-phosphosulfate
- HPLC
- high-performance liquid chromatography
- PCR
- polymerase chain reaction
- RQ
- relative quantification
- HBSS
- Hank's balanced salt solution
- BSP
- bromosulfophthalein
- DHEAS
- dehydroepiandrosterone-3-sulfate
- SN-38
- 7-ethyl-10-hydroxy-camptothecin.
- Received December 2, 2009.
- Accepted April 1, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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