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

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Abstract

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥ 24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking.

Introduction

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of sequestration by cells. Several cell types react to concentrated amine drugs such as procaine, procainamide, nicotine, atropine, and many others by the formation of multiple and large vacuoles (light microscopy) (Belkin et al., 1962, Finnin et al., 1969, Morissette et al., 2004 and literature cited herein). This occurs at fairly large concentrations (10 4–10 2 M) that are of toxicological interest for many but not all drugs; the clinically used concentrations of local anesthetics and of anti-wrinkle “cosmeceutical” agents like dimethylaminoethanol (Morissette et al., 2007b) fall into this range, as well as that of some α-adrenoceptor agonists (e.g., phenylephrine) used locally as mydriatic agents or decongestants (Morissette et al., 2007a). Nonmedicinal cationic compounds, such as the tertiary amine triethylamine, also produce this morphologic change efficiently, but not quaternary amines such as tetraethylammonium (Morissette et al., 2004), suggesting that the uncharged form of the organic amines is the one that enters cells.

Recent investigations have supported the central role of vacuolar (V)-ATPase in the morphologic response to concentrated cationic drugs (Morissette et al., 2004, Morissette et al., 2005, Morissette et al., 2007a, Morissette et al., 2007b): the specific V-ATPase inhibitor bafilomycin A1 completely prevented the cell vacuolization induced by pharmacologically diverse amine drugs in the cited studies. It has been suggested that drug concentration into acidic vacuoles is due to ion trapping (low rate of retro-diffusion of the positively charged amine at low pH) and that the vacuole enlargement that follows is osmotic (Finnin et al., 1969). However, a formal quantitative approach of this form of transport is missing. A morphological analysis of vacuolated cells suggests that the giant vacuoles originate from the trans-Golgi and perhaps from trans-Golgi-derived organelles that express V-ATPase (e.g., lysosomes, endosomes, and secretory granules; Morissette et al., 2005). Accordingly, there is evidence of secretory pathway inhibition in vacuolar cells (Morissette et al., 2005).

The mitochondrion is another known site of trapping for some cationic drugs. The negative membrane potential of the inner mitochondrial membrane created by ATP synthesis and ion translocation drives the accumulation of highly lipophilic cations in the mitochondrial matrix (Murphy and Smith, 2000, Modica-Napolitano and Aprille, 2001, Weiss et al., 1987). The drugs that are efficiently concentrated in this manner also preferably exhibit a delocalized positive charge, for example an amine function in a resonant system of unsaturated bonds. Other cationic drugs bind to DNA according to different modes (intercalation for the anthracyclines, minor groove binding for the dye Hoechst 33258) and label cell nuclei (Tse and Boger, 2004). These mechanisms may also support cationic drug uptake in intact cells in addition to several membrane transporters, which have been shown to carry cationic drugs of various types: multiple organic cation transporters (OCTs), transporters for choline and others (Allen and Lockman, 2003, Zhang et al., 2006). These molecules are often expressed by cell types and tissues specialized in the handling of xenobiotics or endogenous solutes of special importance (the kidney, liver, placenta…).

Following Moriyama (1996), we have hypothesized that metabolically driven sequestration of cationic drugs into intracellular organelles may support intense drug uptake in intact cell types that are not specialized in the handling of xenobiotics. Thus, a pseudotransport driven by intracellular sources of energy (the mitochondrion, ATP utilized by V-ATPase) or affinity for the large reservoir of nuclear DNA may lead to a large cellular concentration of cationic drugs without the need for membrane transporter. Particularly, little quantitative information exists on drug uptake mediated by V-ATPase-driven ion trapping (intensity, apparent affinity, and morphological correlates).

To follow up previous studies from our laboratory, we have used vascular smooth muscle cells (large, very adherent and amenable to morphological analysis) to model the postulated cationic drug uptake (Morissette et al., 2005); procainamide, an agent previously exploited by us, has been used in the quantitative approach to characterize V-ATPase-mediated uptake. This drug transport has been kinetically and pharmacologically compared with those of rhodamine 6G (a delocalized cation known to be concentrated in mitochondria; Bunting, 1992) and of Hoechst 33258, a well known nuclear dye, to unravel the properties of V-ATPase-mediated drug uptake. We continued the characterization of procainamide-induced vacuolar cytopathology with emphasis on interference with vesicular trafficking and on the significance and fate of these large cellular structures.

Section snippets

Drugs

The synthetic V-ATPase inhibitor, FR 167356 (2,6-dichloro-N-[3-(1-hydroxy-1-methylethyl)-2-methyl-7-benzofuranyl]benzamide) (Niikura et al., 2004), was a generous gift from Astellas Pharma, Osaka, Japan. Bafilomycin A1 was purchased from LC Laboratories (Woburn, MA). All other drugs were obtained from Sigma-Aldrich (St. Louis, MO).

Cells and transfection

The institutional research ethics board approved the anonymous use of human umbilical cord segments obtained after elective cesarean deliveries. All culture surfaces

Uptake of cationic drugs by smooth muscle cells

The human umbilical artery smooth muscle cells actively concentrated the tertiary amine procainamide, as shown by fluorometric determination in cellular extract of washed cells (Fig. 1). The cellular accumulation of the drug was maximal after 2 h of incubation with the 2.5 mM concentration level of procainamide in the culture medium (Fig. 1A). This preceded the morphological response, as more than half of the uptake had taken place at 30 min (nonvacuolar cells) and as the cells were more

Pseudotransport of procainamide

Moriyama (1996) has discussed cellular systems where drugs and toxins are compartmentalized by concentration mechanisms relying on chemiosmotic energy-coupling mechanisms. The V-ATPase and mitochondrial ATP synthase (= F-ATPase) are homologous molecular machines that create or exploit H+ gradients in specific endomembranes, respectively. The V-ATPase is distributed to the trans-Golgi and derived structures including the endosomes, lysosomes, secretory granules and at the plasma membrane

Acknowledgments

This work was supported by a grant from the Canadian Institutes of Health Research (operating grant MOP-74448 to F.M., Canada Graduate Scholarships Doctoral Award to G.M.). This public sponsor had no role in the study design or execution or the decision to submit the paper for publication. We thank Ms. Johanne Bouthillier for the technical help, Drs. Marc Pouliot and Paul Naccache (CHUQ-CHUL) for facilitating the access to imaging and fluorometric equipment. We also thank J. Bonifacino and G.

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