Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: Procainamide-induced autophagic cell vacuolization

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

“…Cells extracts equivalent to 1.6 × 10 6 cells per lane were run on 12% SDS-PAGE and transferred to PVDF membranes. Anti-human LC3B rabbit polyclonal antibodies (Novus; dilution 1:3000) were used to detect the cytosolic form LC3-I and processed particulate form LC3-II in PMNLs treated with quinacrine for 0-4 h (Morissette et al, 2008). derived from the microscopic analysis of fluorescent dextran uptake were averaged for many cells and the effects of cell type was evaluated using the Mann-Whitney test (appropriate for the non-normal distributions found; InStat 3.05 program, GraphPad Software).…”

“…In examined cultured cells, the driving force of this pseudo-transport is provided by vacuolar (V)-ATPase, a proton pump expressed in the trans-Golgi and derived organelles (endosomes, lysosomes, secretory granules). Thus, specific vacuolar (V-)ATPase inhibitors such as bafilomycin A1 have been extensively used to document the cellular capture and retention of amines, such as triethylamine (Et 3 N; Morissette et al, 2005) and drugs from various therapeutic classes that can be considered to be substituted forms of this tertiary amine: in increasing order of lipophilicity, procainamide (Morissette et al, 2008), lidocaine (Bawolak et al, 2010), chloroquine (Zheng et al, 2011), quinacrine 2013) and amiodarone (Stadler et al, 2008;Morissette et al, 2009). The threshold concentration for inducing the retention of amines and the characteristic vacuolar morphology that derives from it decreases with increased lipophilicity because the first step of ion trapping is believed to be simple diffusion of the uncharged form though the plasma and vacuolar membranes.…”

Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

“…Previous morphological investigations mostly based on procainamide effects showed that the subcellular origin of a fraction of the giant vacuoles was the trans-Golgi. 15,16 Mitotic arrest without overt toxicity was also observed in vacuolar cells. [15][16][17][18] The present experiments aim to characterize the cell morphological and toxic responses to local anesthetics relative to the vacuolar-autophagic state.…”

“…15,16 Mitotic arrest without overt toxicity was also observed in vacuolar cells. [15][16][17][18] The present experiments aim to characterize the cell morphological and toxic responses to local anesthetics relative to the vacuolar-autophagic state. Novel questions addressed include the clinical relevance of drug concentrations that cause this response and its time frame, the relationship of the vacuolar response to cytotoxicity, the experimental dissociation of the three postulated steps of the vacuolar response ( Fig.…”

“…1). [11][12][13][14][15] The V-ATPase-dependent trapping of the cationic form of the drugs in acidic vesicles is sequentially followed by their osmotic swelling (Step 1), their macroautophagic envelopment (Step 2), and their maturation with lysosome fusion (Step 3) with progressive acquisition of marker proteins typical of autophagy (e.g., LC3) and late endosomes-lysosomes (e.g., CD63) by vacuoles. Previous morphological investigations mostly based on procainamide effects showed that the subcellular origin of a fraction of the giant vacuoles was the trans-Golgi.…”