Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy.
One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.
Breen
Christopher M
CM
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Sykes
Destiny B
DB
Baehr
Carsten
C
Fricker
Gert
G
Miller
David S
DS
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001993:Bronchodilator Agents / Q000494:pharmacology; D002831:Choroid Plexus / Q000378:metabolism*; D004077:Digoxin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005452:Fluoresceins / Q000493:pharmacokinetics*; D008297:Male; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D018613:Microscopy, Confocal / Q000379:methods*; D027361:Organic Anion Transporters / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D001993:Bronchodilator Agents; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D027361:Organic Anion Transporters; D011422:Propionates; D011804:Quinolines; C103864:fluorescein-methotrexate; C059141:verlukast; D004077:Digoxin; D008727:Methotrexate