Formation and rapid export of the monochlorobimane-glutathione conjugate in cultured rat astrocytes.
Monochlorobimane (MCB) is often used to visualize glutathione (GSH) levels in cultured cells, since it is quickly converted to a fluorescent GSH conjugate (GS-MCB). To test for consequences of MCB application on the GSH metabolism of astrocytes, we have studied rat astrocyte-rich primary cultures as model system. MCB caused a concentration dependent rapid decrease in the cellular GSH content. Simultaneously, a transient accumulation of GS-MCB in the cells was observed with a maximal content 5 min after MCB application. The cellular accumulation was followed by a rapid release of GS-MCB into the medium with a maximal initial export rate of 27.9 +/- 6.5 nmol h(-1) mg protein(-1). Transporters of the family of multidrug resistance proteins (Mrps) are likely to be involved in this export, since the Mrp inhibitor MK571 lowered the export rate by 60%. These data demonstrate that, due to its rapid export from astrocytes, GS-MCB is only under well-defined conditions a reliable indicator of the cellular GSH concentration and that MK571 can be used to maintain maximal GS-MCB levels in astrocytes.
Waak
Jens
J
Center for Biomolecular Interactions Bremen, University of Bremen, PO Box 33 04 40, D-28334, Bremen, Germany.
Dringen
Ralf
R
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D018432:Drug Resistance, Multiple; D005456:Fluorescent Dyes; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011422:Propionates / Q000494:pharmacology; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D005456:Fluorescent Dyes; D011422:Propionates; D011720:Pyrazoles; D011804:Quinolines; C059141:verlukast; C059597:monochlorobimane; D005978:Glutathione