Organic anion transporter 6 (Slc22a20) specificity and Sertoli cell-specific expression provide new insight on potential endogenous roles.

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 (K(i)) values of 3.0 +/- 0.5, 48.9 +/- 10.3, 61.4 +/- 7.1, 59.9 +/- 4.9, and 38.8 +/- 3.1 microM, respectively. Comparison to K(i) 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 microM, 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.

Transport of estrone sulfate by the novel organic anion transporter Oat6 (Slc22a20).

Recently, a novel Slc22 gene family member expressed in murine olfactory mucosa was identified and based on sequence homology proposed to be an organic anion transporter [Oat6 (Slc22a20); J. C. Monte, M. A. Nagle, S. A. Eraly, and S. K. Nigam. Biochem Biophys Res Commun 323: 429-436, 2004]. However, no functional data for Oat6 was reported. In the present study, we demonstrate that murine Oat6 mediates the inhibitable transport of estrone sulfate using both Xenopus oocyte expression assay and Chinese hamster ovary (CHO) cells stably transfected with mOat6 (CHO-mOat6). Uptake was virtually eliminated by probenecid and the anionic herbicide 2,4-dichlorophenoxyacetate. The organic anions ochratoxin A, salicylate, penicillin G, p-aminohippurate, and urate inhibited mOat6-mediated accumulation to varying degrees. Transport of estrone sulfate by mOat6 was demonstrated to be saturable, and K(m) estimates of 109.8 +/- 22.6 microM in oocytes and 44.8 +/- 7.3 microM in CHO-mOat6 cells were obtained. Inhibitory constants for 2,4-dichlorophenoxyacetate (15.7 +/- 2.0 microM), salicylate (49.0 +/- 4.4 microM), probenecid (8.3 +/- 2.5 microM), and penicillin G (1,450 +/- 480 microM) were also determined. Accumulation of estrone sulfate mediated by mOat6 was significantly trans-stimulated by glutarate, indicating that mOat6 functions as an organic anion/dicarboxylate exchanger. These data demonstrate for the first time that the novel murine gene Oat6 (Slc22a20) encodes a functional organic anion transporter and mOat6 is indeed the newest member of the OAT gene family.