Abstract
The proton pump vacuolar (V)-ATPase is the driving force that mediates the concentration of cationic drugs (weak bases) in the late endosome-lysosome continuum; secondary cell reactions include the protracted transformation of enlarged vacuoles into autophagosomes. We used the inherently fluorescent tertiary amine quinacrine in murine models to further assess the accumulation and signaling associated with cation trapping. Primary fibroblasts concentrate quinacrine ∼5,000-fold from their culture medium (KM 9.8 µM; transport studies). The drug is present in perinuclear granules that are mostly positive for Rab7 and LAMP1 (microscopy). Both drug uptake and retention are extensively inhibited by treatments with the V-ATPase inhibitor bafilomycin A1. The H+ ionophore monensin also prevented quinacrine concentration by fibroblasts. However, inhibition of plasma membrane transporters or of the autophagic process with spautin-1 did not alter quinacrine transport parameters. Ancillary experiments did not support that low micromolar concentrations of quinacrine are substrates for organic cation transporters-1 to -3 or P-glycoprotein. The secondary autophagy induced by quinacrine in cells may derive from the accumulation of incompetent autophagolysosomes, as judged from the accumulation of p62/SQSTM1 and LC3 II (immunoblots). Accordingly, protracted lysosomogenesis is evidenced by increased expression of LAMP1 and LAMP2 in quinacrine-treated fibroblasts (48 h, immunoblots), a response that follows the nuclear translocation of the lysosomal genesis transcription factor TFEB and upregulation of LAMP1 and −2 mRNAs (24 h). Quinacrine administration to live mice evidenced variable distribution to various organs and heterogeneous accumulation within the lung (stereo-microscopy, extraction). Dose-dependent in vivo autophagic and lysosomal accumulation was observed in the lung (immunoblots). No evidence has been found for transport or extrusion mechanisms modulating the cellular uptake of micromolar quinacrine at the plasma membrane level. As shown in vitro and in vivo, V-ATPase-mediated cation sequestration is associated, above a certain threshold, to autophagic flux inhibition and feed-back lysosomogenesis.
Highlights
The concentration of cationic drugs in acidic cell compartments is well documented, at least in cultured cells (De Duve et al, 1974; Marceau et al, 2012) and freshly isolated peripheral blood leukocytes (Roy et al, 2013)
Quinacrine has 2 protonable functions and high lipophilicity; considerations relevant for its use in vivo are its low toxicity and the fact that its major metabolite, a desethyl secondary amine (Huang et al, 2006), keeps its tropism for acidic organelles, high lipophilicity and fluorescence
The specific inhibitor of all mammalian V-ATPase isoforms, bafilomycin A1, extensively reduced the > thousand-fold concentration of this drug in murine fibroblasts (Figs. 1, 3 and 4); this applied to HEK 293a cells (Fig. S5)
Summary
The concentration of cationic drugs (weak bases) in acidic cell compartments is well documented, at least in cultured cells (De Duve et al, 1974; Marceau et al, 2012) and freshly isolated peripheral blood leukocytes (Roy et al, 2013). A general model is emerging: the proton pump vacuolar (V)-ATPase is the driving force that mediates the concentration and retention of such amines, mainly in the late endosome-lysosome continuum. The ion trapping theory holds that amine drugs become protonated and incapable of retro-diffusion in organelles that are constantly acidified; secondary cell reactions include the osmotic expansion of vacuoles and their protracted (1–4 h) transformation into autophagosomes (Marceau et al, 2012). While the ion trapping model of the cellular concentration of organic amines has been verified in a number of cell types, many issues in this model remain uncertain or not widely verified:. There is limited evidence for the latter process in cells treated with desmethylclomipramine or chloroquine, based on the accumulation of p62/SQSTM1, a protein cleared by autophagy (Iwai-Kanai et al, 2008; Rossi et al, 2009; Sheen et al, 2011), or based on the vacuolar accumulation of tagged recombinant LC3 in cells treated with norephedrine (Funakoshi et al, 2013)
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