Abstract
Organic anion transporter 6 (Oat6; Slc22a20), a member of the OAT family, was demonstrated previously to mediate the transport of organic anions (Am J Physiol Renal Physiol 291:F314–F321, 2006). In the present study, we sought to further delineate the function of murine Oat6 (mOat6) by analyzing the effect of select organic anions on mOat6-mediated transport by using a Chinese hamster ovary (CHO) cell line stably expressing mOat6 (CHO-mOat6). When examined, kinetic analysis demonstrated that the mechanism of inhibition of mOat6 and mOat3 was competitive. Homovanillic acid, 5-hydroxyindole acetic acid, 2,4-dihydroxyphenylacetate, hippurate, and dehydroepiandrosterone sulfate (DHEAS) each significantly reduced mOat6 activity with inhibitory constant (Ki) values of 3.0 ± 0.5, 48.9 ± 10.3, 61.4 ± 7.1, 59.9 ± 4.9, and 38.8 ± 3.1 μM, respectively. Comparison to Ki values determined for mOat3 (67.8 ± 7.2, 134.5 ± 27.0, 346.7 ± 97.9, 79.3 ± 4.0, and 3.8 ± 1.1 μM, respectively) revealed that there are significant differences in compound affinity between each transporter. Fluoroquinolone antimicrobials and reduced folates were without effect on mOat6-mediated uptake. Investigation of testicular cell type-specific expression of mOat6 by laser capture microdissection and quantitative polymerase chain reaction revealed significant mRNA expression in Sertoli cells, but not in Leydig cells or spermatids. Overall, these data should aid further refinements to the interpretation and modeling of the in vivo disposition of OAT substrates. Specifically, expression in Sertoli cells suggests Oat6 may be an important determinant of blood-testis barrier function, with Oat6-mediated transport of estrone sulfate and DHEAS possibly representing a critical step in the maintenance of testicular steroidogenesis.
Footnotes
This research was supported in part by a National Institutes of Health Training Grant in Environmental Stress Signaling [Grant T32ES012878] (to G.W.S.) and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01DK067216] (to D.H.S.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.168765.
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ABBREVIATIONS:
- OAT
- organic anion transporter
- mOat
- murine Oat
- BEB
- blood-epididymal barrier
- BTB
- blood-testis barrier
- 5-CH3-THF
- 5-methyltetrahydrofolate
- CHO
- Chinese hamster ovary
- 2,4-D
- 2,4-dichlorophenoxyacetate
- DHEAS
- dehydroepiandrosterone sulfate
- DOPAC
- 2,4-dihydroxyphenylacetic acid
- ES
- estrone sulfate
- HA
- hippuric acid
- 5-HIAA
- 5-hydroxyindoleacetic acid
- HVA
- homovanillic acid
- PAH
- para-aminohippurate
- QPCR
- real-time quantitative polymerase chain reaction
- SLC
- solute carrier
- LCM
- laser capture microdissection
- Gapdh
- glyceraldehyde 3-phosphate dehydrogenase
- FRT
- flippase recognition target.
- Received March 29, 2010.
- Accepted June 1, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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