Abstract:We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) 3 H]DHEAS across the BBB (118 l/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 l/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [3 H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K m of 34.4 M and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [ 3 H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [ 3 H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB. Key Words: Blood-brain barrier-Dehydroepiandrosterone sulfate -Efflux transport-Organic anion transporting polypeptide 2-Conditionally immortalized mouse brain capillary endothelial cell line.

The cerebrospinal fluid-to-blood efflux transport of estrone-3-sulfate (E 1 S) via the blood-cerebrospinal fluid barrier (BCSFB) may play an important role in regulating E 1 S levels in the brain. Here, we investigated the efflux transport of E 1 S at the BCSFB using conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB) and identified the responsible transporter. The [ 3 H]E 1 S uptake by TR-CSFB cells was composed of saturable and nonsaturable components, and the K m and V max values of the saturable component were determined to be 16.8 Ϯ 5.1 M and 12.3 Ϯ 2.3 pmol/min/mg of protein, respectively. [ 3 H]E 1 S uptake was inhibited by probenecid, cholate, taurocholate, sulfobromophthalein, dehydroepiandrosterone sulfate, triiodothyronine, thyroxin, and digoxin but not by p-aminohippuric acid, ␥-aminobutyric acid, or methotrexate, suggesting the involvement of organic anion transporting polypeptide (oatp) in the uptake. Reverse transcription-polymerase chain reaction analysis revealed that oatp3 was expressed in TR-CSFB cells and isolated rat choroid plexus, although oatp1 was not detected in either. Xenopus laevis oocytes expressing oatp3 exhibited [ 3 H]E 1 S uptake activity with a K m of 8.09 Ϯ 2.83 M and V max of 8.02 Ϯ 0.87 pmol/h/oocyte. Moreover, oatp3 is localized at the brush-border membrane of choroid plexus epithelial cells. These results suggest that oatp3 is involved in the E 1 S efflux transport at the BCSFB.

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E 2 , between extra-and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells. Keywords: blood-brain barrier, choroid plexus, hypothalamus, olfactory bulb, organic anion transporting polypeptide 3. Organic anion transporting polypeptide 3 (oatp3; Slc21a7) mediates sodium-independent transport of a wide variety of amphipathic organic compounds, including opioid peptides, neurosteroid conjugates, thyroid hormones (THs), bile acids and drugs, such as fexofenadine (Abe et al. 1998;Dresser et al. 2002;Hagenbuch and Meier 2003). Since many of these substrates are active in the CNS, oatp3 may play a role in the control of CNS functions.THs play an important role in normal CNS development and maturation. The expression of TH receptors was detected in adult brain (Puymirat et al. 1991), and hypothyroidism induces changes in the neuronal morphology of adult rat brain (Ruiz-Marcos et al. 1980) and affects memory in human patients (Burmeister et al. 2001). THs interact with nuclear TH receptors to express their effects. Although it was believed that THs enter target cells by passive diffusion, the recent reports have demonstrated that their cellular uptake by cells, including neurons and astrocytes, is carrier-mediated (Francon et al. 1989;Chantoux et al. 1995;Hennemann et al. 2001). Although roles of oatp family members in neural cells have not been considered yet, it is conceivable that oatps mediate TH uptake of the target cells in the brain.Expression of oatp3 mRNA in rat brain has been detected by means of RNase protection assay and RT-PCR analysis (Walters et al. 2000;Ohtsuki et al. 2003). Since oatp3 mediates transport of THs, such as triiodothyronine (T 3 ) and thyroxine (T 4 ) (Abe et al. 1998), we hypothesize that oatp3 is expressed at the neural cells and mediates their TH uptake. Received January 13, 2004; revised manuscript received March 19, 2004; accepted April 8, 2004.Address correspondence ...

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