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Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17beta-glucuronide (E(2)17betaG). Intriguingly, published data suggest that E(2)17betaG inhibition of Oatp1c1-mediated T(4) transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T(4) and E(2)17betaG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T(4) and E(2)17betaG concentrations (100 pm to 300 nm and 27 nm to 200 mum, respectively). Eadie-Hofstee plots derived from these detailed T(4) and E(2)17betaG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T(4) and E(2)17betaG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T(4) and E(2)17betaG. T(4) and E(2)17betaG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E(2)17betaG transport more strongly than T(4) transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

Organic anion transporting polypeptide (Oatp) 1c1 is a high-affinity T4 transporter with narrow substrate specificity expressed at the blood-brain barrier. A transport model using cells overexpressing Oatp1c1 was created to identify novel Oatp1c1 substrates and inhibitors. Rat Oatp1c1 was cloned and stably expressed in human embryonic kidney 293 cells. Oatp1c1-transfected human embryonic kidney 293 cells transported 125I-labeled T4 in a time-dependent manner that was completely abolished in the presence of excess unlabeled T4. Next, various compounds, including inhibitors of thyroid hormone uptake, were screened for inhibitory effects on Oatp1c1-mediated T4 uptake. Phenytoin (64%), indocyanine green (17%), fenamic acid (68%), diclofenac (51%), and meclofenamic acid (33%) all reduced T4 uptake by Oatp1c1 when assayed at concentrations of 10 μM. Dose-response assays for the fenamic acids, iopanoic acid, indocyanine green, and phenytoin revealed IC50 values for Oatp1c1 T4 uptake below or near the blood plasma levels after therapeutic doses. Further kinetic assays and reciprocal plot analyses demonstrated that the fenamic acid diclofenac inhibited in a competitive manner. Finally, microvessels were isolated from adult rat brain and assessed for T4 uptake. Ten micromolar of fenamate concentrations inhibited T4 microvessel uptake with a similar hierarchical inhibition profile [fenamic acid (43%), diclofenac (78%), and meclofenamic acid (85%)], as observed for Oatp1c1 transfected cells. Oatp1c1 is expressed luminally and abluminally in the blood-brain barrier endothelial cell, and exhibits bidirectional transport capabilities. Together, these data suggest that Oatp1c1 transports fenamates into, and perhaps across, brain barrier cells. The fenamate class of nonsteroidal anti-inflammatory drugs is transported by organic anion transporting polypeptides and competitively inhibits thyroxine transport in brain microvessels.

Organic anion-transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood-brain barrier endothelial cells and blood-cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics.