Vacuolar ATPase-mediated cellular concentration and retention of quinacrine: a model for the distribution of lipophilic cationic drugs to autophagic vacuoles.
The antiprotozoal agent quinacrine is a lipophilic cationic drug highly distributed to tissues. It has been used in the present experiments to examine whether the vacuolar and autophagic cytopathology induced by organic amines is independent from the therapeutic class. Furthermore, we tested the presence of the concentrated cationic drug itself in the enlarged vacuoles by exploiting the intense green fluorescence of quinacrine. Finally, the influence of lipophilicity on the apparent affinity of amine pseudotransport has been addressed by comparing quinacrine to another substituted triethylamine, procainamide. Quinacrine was concentration-dependently taken up by human smooth muscle cells (cytosolic granular-vacuolar morphology at and above 25 nM; in cell extracts, uptake nearly maximal in 2 h, apparent K(m) of 8.7 microM). The vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine uptake by cells or released the cell-associated drug in preloaded cells. The lipidated (II) form of microtubule-associated protein light chain 3 accumulated at and above a quinacrine concentration of 2.5 microM (4 h), indicating the conserved macroautophagic nature of the vacuolar cytopathology, although vacuole size was modest. The enlarged vacuoles containing quinacrine excluded cherry fluorescent protein; many vacuoles were lined with cherry fluorescent protein-conjugated Rab7, a GTPase associated with late endosomes/lysosomes. Taken together, these results are compatible with the transition of quinacrine-concentrating vacuoles toward an autophagolysosome identity. Quinacrine is concentrated in cells via V-ATPase-mediated ion trapping with an apparent affinity approximately 500-fold higher than that of the less lipophilic drug procainamide, and, despite the small size of ensuing vacuoles, the macroautophagic signature of this cytopathology was observed.
Marceau
François
F
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, and Department of Medicine, Université Laval, Québec, Canada G1V 4G2. francois.marceau@crchul.ulaval.ca
Bawolak
Marie-Thérèse
MT
Bouthillier
Johanne
J
Morissette
Guillaume
G
D000981:Antiprotozoal Agents / Q000378:metabolism* / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects; D001692:Biological Transport; D002412:Cations; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D018942:Macrolides / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D053141:Organelle Size; D011342:Procainamide / Q000378:metabolism; D011796:Quinacrine / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D000981:Antiprotozoal Agents; D002412:Cations; D004791:Enzyme Inhibitors; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D011796:Quinacrine; D011342:Procainamide