Short Communication 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 Km of 8.7 M). 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 M (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 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. It has been proposed a long time ago that the particular vacuolar cytopathology induced by cationic drugs is due to ion trapping, with the protonation of the drug at low pH in acidic cell organelles, decreased rate of retrodiffusion, and subsequent osmotic swelling of the vacuoles (de Duve et al., 1974). Recent progress in this research area supports the fact that the proton pump vacuolar (V)-ATPase is necessary for this form of cationic drug sequestration in intact cultured cells (a form of pseudotransport (Morissette et al., 2005, 2008b). It was once believed that vacuolar cytopathology was of lysosomal origin, and the amines that caused it were dubbed “lysosomotropic”; however, this explanation is an oversimplification because many of the giant vacuoles originate from the trans-Golgi, which also expresses V-ATPase (Morissette et al., 2005, 2008b). On the other hand, it was recently discovered that the giant vacuoles induced by cationic drugs rapidly become macroautophagic, being labeled with the membrane-bound (lipidated) form of the autophagic effector microtubuleassociated protein light chain 3 [(LC3) II], and the vacuoles acquire late endosome/lysosome markers, presumably due to lysosomal fusion to autophagosomes (Morissette et al., 2005, 2008b). The intense concentration of the tertiary amine procainamide in human vascular smooth muscle has been the primary model exploited to reach these conclusions, exhibited an apparent Km of 4.7 mM, was abolished by chemically unrelated V-ATPase inhibitors (bafilomycin A1 or FR 167356), and preceded the formation of multiple large and clear vacuoles (Morissette et al., 2008b). The postulated first step of this form of sequestration is the crossing of plasma and vacuolar membranes by simple diffusion, a process that is inhibited for procainamide by acidifying the extracellular fluid (Morissette et al., 2008b) and which should be greater for drugs with increased lipophilicity. Consistent with this idea, the antiarrhythmic agent amiodarone (logP 7.2), which can be considered as a substituted triethylamine similar to the more hydrophilic agent procainamide (logP 1.13), was concentrated in cell vacuoles and activated LC3 processing (formation of LC3 II) at 5 to 20 M (Morissette et al., 2009). Accumulation of macroautophagic vacuoles has been observed in the skin of a patient with amiodarone-induced skin discoloration (Morissette et al., 2009), a side effect caused by drug deposition in tissues (Ammoury et al., 2008). The antiprotozoal agent quinacrine can also be considered a substituted triethylamine, and it has an intermediate lipophilicity (logP 5.67), two protonable functions (pKa 10.3 and 7.7), an intense green fluorescence, and occasionally causes skin discoloration upon chronic dosing (Zuehlke et al., 1981), similar to amiodarone. Quinacrine has been used in the present experiments to further clarify the following: This study was supported by the Canadian Institutes of Health Research [Grant MOP-74448]; Canada Graduate Scholarships Doctoral Award (to G.M.); and Fonds de la Recherche en Santé du Québec, QC, Canada (Studentship Award to