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Ex Vivo Fluorometric Analysis of Efflux Transporters in Rat Choroid Plexus
Author(s) -
Ocheltree Scott Matthew,
Hawkins Brian T.,
Egleton Richard D.,
Davis Thomas P.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1143
Subject(s) - choroid plexus , efflux , rhodamine 123 , chemistry , multidrug resistance associated protein 2 , in vivo , transporter , apical membrane , ex vivo , pharmacology , biochemistry , biophysics , microbiology and biotechnology , atp binding cassette transporter , biology , in vitro , membrane , endocrinology , central nervous system , multiple drug resistance , antibiotics , gene
The strategic localization of efflux transporters (e.g., P‐gp, MRP1, BCRP) to the apical and basolateral membranes of the choroid plexus suggests that they play important roles in the disposition of endo‐ and xenobiotics in the brain. We developed a rapid, sensitive and nondestructive fluorometric method to study the functional modulation of efflux transporters at the choroid plexus during disease (i.e., inflammatory pain). Ex vivo whole‐tissue rat choroid plexus uptake studies were performed with rhodamine 123 in bicarbonate artificial cerebrospinal fluid (pH 7.4) under various experimental conditions. The fluorescent uptake of each sample was determined by a GENios ® fluorescence plate reader (485 nm excitation and 535 nm emission filters). Cell uptake (nmol/g wet tissue) was calculated from a standard curve of rhodamine 123. The limit of detection for rhodamine 123 was 47 fmols. Under physiological conditions rhodamine 123 (60 nM) accumulation in choroid plexus was linear up to 45 minutes. However, rhodamine 123 accumulation was reduced by 75% at 4°C (p<0.001). Inhibition studies performed with 60 μM novobiocin (specific inhibitor of BCRP) reduced uptake by 24% (p<0.05). This analytical method offers advantages over quantification with radiolabeled substrates such as: rapid acquisition of data, increased sensitivity, non‐destructive, thereby allowing preparation of cellular protein for western blot analysis. This method has proven to be a simple and efficient means for characterizing the functional significance of drug efflux transporters in choroid plexus. Support: NINDS R01NS42652, NIDDK R01DK065003, NIH T32 HL07249