Chloroquine (CQ), the most widely used antimalarial drug, is an acidotropic agent (De Duve, 1983) which accumulates to high levels in malaria-infected erythrocytes. A possible site of accumulation of the drug, the parasite's food vacuole, has been implicated in the mode of action of CQ. We have defined the various compartments of Plasmodium falciparumparasitized human erythrocytes in tenns of their pH and capacity to accumulate bases. The host cell and the parasite cytosols were differentially labeled in situ with pH-sensitive fluorescein, and the parasite food vacuole was revealed by tareting fluoresceinated dextran via endocytosis. The pH of the various compartments obtained from fluorescence excitation spectra were 6.9 for the cytosol of normal and infected erythrocytes and 5.2 for the parasite food vacuole. Determination of CQ and methylamine accumulation in infected erythrocytes, in conjunction with morphometric determination of the relative sizes of the various cellular compartments, provided an independent assessment of the vacuolar pH, yielding a value of 5.0-5.2. Perturbation of the proton gradient, either by lowering extracellular pH or by alkaliniation of the food vacuole with NH4CI or monensin, resulted in a concomitant and reversible decrease in accumulation of the probe. We conclude that drug accumulation in malariainfected erythrocytes can be fully accounted for by the steady-state proton gradients across the barriers delineating the various cellular compartments and the acidotropic properties of the drug.Key words: malaria/Plasmodium falciparum/intracellular pH/food vacuole/chloroquine/fluorescence Introducdton Chloroquine (CQ) is a widely used antimalarial drug. The development of resistance to this compound is one of the major reasons for the world wide resurgence of the disease during recent years (Peters, 1982). The mode of action of the drug has not been elucidated hitherto, let alone the mechanisms responsible for drug resistance. A major factor in the antimalarial action of the drug has been attributed to the ability of CQ-sensitive parasites to accumulate relatively high levels of the drug (Macomber et al., 1966). It has been proposed that this accumulation results from binding to a putative intraparasitic receptor, ferriprotoporphyrin IX (FP) with the consequent formation of a membrane lytic complex (Fitch, 1983). However, free FP has never been detected in malaria-infected red blood cells. Alternatively, by analogy with the lysosomotropic effects of weak bases on animal cells (Poole and Okhuma, 1981), it has been postulated that CQ accumulates to high levels in the food vacuoles of malaria parasites, causing their concomitant alkalinization (Homewood et al., 1972).In this study we defined the role of transmembrane proton gradients as the determining factor in CQ accumulation by Plasmodiumfalciparum-infected human erythrocytes (RBC). We have done this with the aid of fluorescent probes and labeling procedures which allowed us to gain access to various intracellular compartments of the in...

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the lattereither by changing the external pH or by adding the acidotropic agent NH4Cl-led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 x 10-7 M, 1.8 x 10-7 M, and 3.3 x 10-7 M, respectively. Marked resistance to CQ (IC50 = 9.8 x 10-7 M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.The development of resistance of malarial parasites to the drug chloroquine (CQ), for many years the most popular and efficient antimalarial drug, has been of growing concern (1). However, the factors responsible for drug resistance have not been identified nor has the mode of CQ action been fully elucidated. The antimalarial effect of the drug has been attributed to the ability of CQ-sensitive parasites to accumulate relatively high levels of the drug (2, 3). It has been proposed that CQ accumulation results from its binding to a putative intraparasitic receptor, ferriprotoporphyrin IX (FeP), with consequent formation of a membrane lytic agent (4). Alternatively, in analogy with the lysosomotropic effects of weak bases on animal cells (5), it was postulated that CQ accumulates to high levels in the acidic food vacuoles of malaria parasites, causing their alkalinization and interfering with vital cellular processes (6).This latter hypothesis rests on the idea that the food vacuole of an intraerythrocytic malaria parasite is an acidic compartment (7,8) whose pH is maintained by metabolic input (9) and in which CQ accumulates by virtue of its weak base properties, following transmembrane proton gradients. This has recently been demonstrated by fluorescence measurements of intravacuolar pH and determination of CQ distribution as a function of established pH gradients and their dissipation by various agents (10). However, at therapeutic levels, the effect of CQ on intravacuolar pH, as determined by CQ and methylamine distribution, was minor, thus casting doubt on the validity of vacuolar alkalinization per se as a major factor in CQ-mediated inhibition of parasite development.The mode of action of CQ and the mechanism of drug resistance in human malaria have been assessed...

The intraerythrocytic human malarial parasite Plasmodium falciparum produces lactate at a rate that exceeds the maximal capacity of the normal red cell membrane to transport lactate. In order to establish how the infected cell removes this excess lactate, the transport of lactate across the host cell and the parasite membranes has been investigated. Transport of radiolabeled L-lactate across the host cell membrane was shown to increase ca. 600-fold compared to uninfected erythrocytes. It showed no saturation with [L-lactate] and was inhibited by inhibitors of the monocarboxylate carrier, cinnamic acid derivatives (CADs), but not by the SH-reagent p-chloromercuriphenyl sulfonic acid (PCMBS). These results suggest that L-lactate is translocated through CAD-inhibitable new pathways induced in the host cell membrane by parasite activity, probably by diffusion of the acid form and through a modified native monocarboxylate:H+ symporter. Continuous monitoring of extracellular pH changes occurring upon suspension of infected cells in isoosmotic Na-lactate solutions indicates that part of the lactate egress is mediated by anionic exchange through the constitutive, but modified, anion exchanger. The transport of L-lactate across the parasite membrane is rapid, nonsaturating, and insensitive to either CADs or PCMBS, or to the presence of pyruvate. L-lactate uptake increased transiently when external pH was lowered and decreased when delta pH was dissipated by the protonophore carbonylcyanide m-chlorophenyl hydrazone (CCCP). These results are compatible with L-lactate crossing the parasite membrane either as the undissociated acid or by means of a novel type of lactate-/H+ symport.

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