ATPase activity of purified plasma membranes and digestive vacuoles from Plasmodium falciparum

Molecular & Biochemical Parasitology 141 (2005) 49–56

ATPase activity of purified plasma membranes and digestive vacuoles from Plasmodium falciparum Laurence M. Elandalloussi 1 , Bronwen Adams 2 , Peter J. Smith ∗ Department of Pharmacology, University of Cape Town, Medical School, Observatory 7925, South Africa Received 1 October 2004; received in revised form 31 January 2005; accepted 3 February 2005

Abstract The ATPase activity of the human malaria parasite, Plasmodium falciparum was investigated using two experimental systems, (i) digestive vacuoles, and (ii) purified plasma membranes isolated from a chloroquine-sensitive and a chloroquine-resistant strain. No correlation between the level of ATPase activity and chloroquine sensitivity could be detected. In both systems, the ATPase activity of the chloroquine-resistant and -sensitive strain was decreased in the presence of the P-glycoprotein inhibitor vanadate. Susceptibility to inhibition by vanadate together with the lack of effect of ouabain implies a P-type ATPase activity in the plasma membrane. Furthermore, the inhibition of Fac8 ATPase activity by oligomycin both in the digestive vacuoles and the plasma membranes would be consistent with higher levels of Pgh1 in Fac8. Our data are consistent with the presence of a V-type H+ -ATPase in the parasite food vacuole. Bafilomycin A1 and N-ethylmaleimide decreased the vacuolar ATPase activity in both chloroquine-resistant and -sensitive strains. Interestingly, a 30% decrease was observed between the ATPase activity of plasma membranes isolated from Fac8 and D10 in the presence of bafilomycin A1, suggesting the presence of a V-type ATPase in D10 plasma membrane that is underexpressed or altered in the plasma membrane of the chloroquine-resistant Fac8. The chemosensitisers tested had no effect on the ATPase activity of chloroquine-resistant P. falciparum in both systems suggesting that their activity is not mediated through an ATP-dependent mechanism. No effect was observed on the vacuolar ATPase activity in the presence of the antimalarials tested indicating that an ATP-dependent transport has not been activated. © 2005 Elsevier B.V. All rights reserved. Keywords: Plasmodium falciparum; ATPase; Chloroquine; Antimalarial drug; Chemoreversal; Membrane

1. Introduction The biochemical basis of chloroquine resistance is still undecided. However, chloroquine is concentrated in the acidic vacuole of the parasite and it is clear that resistant parasites accumulate less chloroquine than sensitive isolates [1]. Current

Abbreviations: ATP, adenosine 5 triphosphate; ATPase, adenosine triphosphatase; MDR, multidrug resistant; NEM, N-ethylmaleimide ∗ Corresponding author. Tel.: +27 21 406 6289; fax: +27 21 448 1989. E-mail address: [email protected] (P.J. Smith). 1 Present address: Centro d’Aquicultura–IRTA, 43540 Sant Carles de la Rapita, Spain. 2 Present address: 12735 Twinbrook Parkway Room 3E-10C, Rockville, MD 20852, USA. 0166-6851/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2005.02.001

hypotheses to explain chloroquine resistance include modification in drug transport [2], altered binding of chloroquine to the heme [1] and changes in digestive vacuole pH [3,4]. A number of candidate genes in Plasmodium falciparum have been proposed to be involved in chloroquine resistance, each concerned in membrane transport. Most notably, chloroquine resistance has been associated in vitro with point mutations in two genes, pfcrt and pfmdr 1, which encode the P. falciparum digestive-vacuole transmembrane proteins PfCRT [5] and Pgh1 [6], respectively. The PfCRT protein resides within the digestive vacuole membrane [5] and has an hydropathy plot suggestive of a transport function. Hence, models wherein mutant PfCRT provokes altered ionic equilibrium across the vacuolar membrane are attractive [7]. A V-type ATPase [8] and a P-glycoprotein homologue (Pgh1) [9] have been re-

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ported to be located in the food vacuole and the plasma membrane of P. falciparum and have been identified as potential sites involved in the mechanism of chloroquine-resistance. V-type ATPases were first believed to be confined on the membrane of intracellular vacuoles. However, they have been since then described in the plasma membrane of various cell types [10]. Interestingly, the protozoan parasites Trypanosoma cruzi [11] and Toxoplasma gondii [12], two apicomplexan-like the malaria parasite, have been shown to possess a V-type H+ -ATPase located in the plasma membrane and in intracellular vacuoles. Studies based on the effect of the specific V-H+ -ATPase inhibitor bafilomycin A1 suggested that this V-ATPase is involved in the regulation of pH in T. cruzi [11] and T. gondii [12]. Similarly, P. falciparum have been shown to possess a bafilomycin A1-sensitive V-type ATPase [13]. Two subunits of the Plasmodium V-ATPase have been cloned [8,14] and subcellular localisation demonstrated that the ATPase is present in the plasma membrane of the parasite, as well as intracellular vesicular structures including the digestive vacuole [15]. Moreover, bafilomycin A1 has been shown to cause acidification of the cytoplasm of the parasite whereas vanadate and amiloride analogues had little effect [16]. These results strongly suggest that a V-ATPase is functionally expressed at the plasma membrane of P. falciparum and pumps protons out of the organism. The predicted amino-acid sequence of Pgh1 shows that it belongs to the superfamily of ATP-binding cassette transporters. In multidrug-resistant cell lines, a Pgp is believed to mediate multidrug resistance by acting as an ATP-dependent exporter of drugs. This model is supported by the observation that transport is dependent on ATP-binding and hydrolysis [17]. It has also been shown that chemosensitisers reverse drug-resistance by increasing accumulation of drug within cells, bind specifically to membrane vesicles from multidrug-resistant (MDR) cells and inhibit vinblastine analogue photoaffinity labelling of the P-170 [18]. Cowman et al. [9] showed that Pgh1 is localized primarily to the membrane of the digestive vacuole of P. falciparum trophozoites and that a small fraction appears to be localized to the parasite membrane. However, photoaffinity studies using a photoreactive analogue of chloroquine failed to demonstrate an interaction of chloroquine with Pgh1 in P. falciparum [19]. Pgh1 is expected to share at least some of these features with other Pgps, although presently only ATP-binding has been demonstrated for Pgh1 [20]. Recently, mutations in Pgh1 have been linked to mefloquine, quinine and halofantrine resistance and these same mutations have been shown not only to influence parasite resistance towards chloroquine in a strain-specific manner but also the level of sensitivity to artemisinin [6]. ATPases can be distinguished according to their relative sensibility to inhibitors. Vanadate is believed to inhibit P-type ATPases by preventing the formation of a phosphoaspartyl intermediate whereas bafilomycin A1 is believed to be a high-affinity inhibitor of V-ATPases. Ouabain is a specific inhibitor of the Na+ /K+ -ATPases while the sulfhydryl reagent

N-ethylmaleimide and oligomycin are known inhibitors of the proton pump and the mitochondrial membrane ATPases, respectively. In this study, we report the effect of P-type and V-type ATPase inhibitors on P. falciparum plasma membrane and vacuolar ATPase activity in order to identify the components implicated in the ATPase activity. We then extend the approach by investigating the effect of chemosensitisers on ATPase activity in both plasma membranes and digestive vacuoles. Finally, the effect of the antimalarials quinine, mefloquine and artemisinin on ATPase activity are examined to determine whether an ATP-dependent transport of these drug occurs either at the plasma membrane level or in the digestive vacuoles and to investigate an eventual regulation of the ATPase activity by the transported substrate in a chloroquine-sensitive and in a chloroquine-resistant strain.

2. Materials and methods 2.1. Cell culture The chloroquine sensitive clone D10 [21], the cultured adapted South African isolates RSA3 and RSA11 and the chloroquine resistant FAC8 [22] and K1 [23] strains of P. falciparum were cultured following a modification of the method of Trager and Jensen [24], as described previously [25]. 2.2. Plasma membrane isolation Parasite plasma membranes were obtained by saponin lysis of the erythrocytes and disruption of the trophozoites by nitrogen decompression as described previously [25]. Trophozoites were purified of erythrocyte membranes by immunoaffinity chromatography using anti-erythrocyte antibodies while the purification of the plasma membranes from contaminants was achieved using a magnetic cell sorting system. 2.3. Digestive vacuole isolation Vacuoles were purified as reported previously [26]. Briefly, late trophozoites were released from erythrocytes by saponin lysis and then homogenised by a combination of hypotonic lysis at low pH and trituration through a fine needle. The resulting crude vacuole preparation was incubated in isotonic buffer containing DnaseI and the purified vacuoles harvested from the bottom of a density gradient using Percoll following high-speed centrifugation. 2.4. Drug dilutions and solvent controls The presence of dimethylsulfoxide, used to solubilise ouabain, oligomycin, bafilomycin A1 and mefloquine at a final concentration of 0.5%, 0.2%, 0.1% and 0.1%, respectively, had no effect on the ATPase activity. Similarly, the

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presence of ethanol used to solubilise progesterone at a final concentration of 0.25% had no effect on the ATPase activity.

Table 1 Chloroquine sensitivity and ATPase activity of parasite plasma membranes and digestive vacuoles isolated from various strains of P. falciparum

2.5. ATPase activity

Strain

The ATPase activity was determined using a colorimetric assay adapted from those of Chifflet et al. [27] and Doige et al. [28]. Enzyme assays were carried out with 50 ␮g of freshly isolated P. falciparum plasma membranes suspended in 450 ␮l of plasma membrane reaction buffer (50 mM Tris–HCl, 0.15 M NH4 Cl, 2 mM MgCl2 , 0.02% NaN3 , pH 7.4) or with 5 × 107 vacuoles in 450 ␮l of vacuole reaction buffer (2 mM MgSO4 , 100 mM KCl, 10 mM NaCl and 25 mM HEPES, pH 7.5). To initiate the reaction 50 ␮l of ATP in the appropriate reaction buffer was added, giving a final concentration of 2 mM ATP. When drugs were used, they were preincubated for 15 min at room temperature prior to the addition of ATP and the appropriate controls were included. After 45 min at 37 ◦ C, the suspensions were centrifuged at 13,000 rpm for 3 min to pellet the membranes/digestive vacuoles. Aliquots of 100 ␮l of the resulting supernatant were transferred to the wells of a 96-well microtitre plate. The reaction was stopped by the addition of 100 ␮l of freshly prepared solution A (6% SDS, 3% l-ascorbic acid, 0.5% ammonium molybdate in 0.25 M sulfuric acid). After 15 min, the phophoammonium molybdate complex formed was stabilised by the addition of 100 ␮l of solution B (2% sodium citrate, 2% sodium arsenite, 2% acetic acid) and the absorbance at 710 nm was measured. 2.6. Protein determinations Protein determinations were performed by the method of Lowry et al. [29]. 2.7. In vitro P. falciparum cytotoxicity assay The IC50s for P. falciparum in the presence of chloroquine was measured by the method developed by Makler et al. [30]. The parasite lactate dehydrogenase assay was used for the evaluation of parasite viability.

3. Results

IC50 a (nM)

D10 28.32 ± 3.53 RSA3 52.46 ± 17.01 Fac8 157.50 ± 55.98 RSA11 252.53 ± 20.79 K1 379.10 ± 34.96

3.25 4.17 11.90 10.05 5.81

± ± ± ± ±

1.75 2.09 1.46 0.79 0.66

ATPase activitya (pmol Pi/min/106 vacuoles) 65.86 ± 5.21 ND 87.81 ± 6.61 68.85 ± 7.73 ND

a Values are means of at least three separate experiments ± standard deviation.

uoles isolated from the sensitive strain D10 displayed an ATPase activity of 65.9 ± 5.2, while those isolated from the resistant strains Fac8 and RSA11 displayed an activity of 87.8 ± 6.6, 68.9 ± 7.7 pmol Pi/min/106 vacuoles, respectively. The ATPase activity in absence of drug of plasma membranes isolated from the sensitive strain D10 and the resistant strains Fac8 and RSA11 was 3.2 ± 1.7, 11.9 ± 1.5 and 10.0 ± 0.8 nmol Pi/min/mg protein, respectively. 3.2. Effect of P-type and V-type ATPase inhibitors To determine their participation in the ATPase activity measured, the effect of P-type and V-type ATPase inhibitors on the ATPase activity of plasma membranes and digestive vacuoles was evaluated. The concentrations of the V-type and P-type ATPase inhibitors tested in this study were similar to those chosen by Choi and Mego [31] to investigate their effect on the Plasmodium food vacuole ATPase activity. The effect of the P-type, vanadate (0.1 mM), olygomycin (50 ␮g/ml) and ouabain (1 mM), and V-type, bafilomycin A1 (1 ␮M) and N-ethylmaleimide (NEM) (2 mM), ATPase inhibitors are summarised in Table 2. Ouabain decreased the ATPase activity of plasma membranes isolated from D10 and Fac8 by 16% and 22%, respectively, indicating that the P. falciparum plasma membrane does not contain an ouabain-sensitive Na+ /K+ -ATPase. Table 2 Effect of the P-type, vanadate (0.1 mM), oligomycin (50 ␮g/ml) and ouabain (1 mM), and V-type, bafilomycin A1 (1 ␮M) and NEM (2 mM), ATPase inhibitors on P. falciparum plasma membranes and vacuolar ATPase activity Inhibitor

3.1. ATPase activity of digestive vacuoles and parasite plasma membranes Hydrolysis of ATP by intact P. falciparum digestive vacuoles and purified plasma membranes was maximal at pH 7.5 and 7.4, respectively and required 2 mM of the divalent cation Mg2+ and 2 mM of the energy source ATP (data not shown). The ATPase activity of digestive vacuoles and plasma membranes isolated from different strains of P. falciparum exhibiting various degrees of chloroquine resistance are shown in Table 1. Purified digestive vac-

ATPase activitya (nmol Pi/min/mg protein)

Percentage of inhibitiona Plasma membranes

Digestive vacuoles

D10

D10

Fac8

Fac8

RSA11

41 ± 23 29 ± 14 14 ± 12 58 ± 13 16 ± 13 22 ± 20

39.8 ± 1.4 51.5 ± 5.5 43.8 ± 2.6 35.8 ± 3.1 60.7 ± 2.9 40.3 ± 4.4 NDb NDb NDb

Bafilomycin A1 47 ± 22 17 ± 14 NEM 32 ± 22 14 ± 20

46.7 ± 4.3 35.3 ± 4.4 51.6 ± 2.8 43.3 ± 4.4 38.7 ± 5.3 40.4 ± 5.9

Vanadate Oligomycin Ouabain

a Values are expressed as percentages of inhibition of ATPase activity compared to controls without drugs and standard deviations from means of at least three separate experiments, each performed in quadruplicate. b ND: not determined.

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ATPase activity of membrane purified from the chloroquinesensitive strain D10. Artemisinin (1 mM) had no effect on the ATPase activity of purified plasma membranes from the chloroquine-resistant and -sensitive strain, respectively.

4. Discussion

Fig. 1. Effect of bafilomycin A1 (1 ␮M), vanadate (100 ␮M) and their combination on vacuolar ATPase activity. Values are expressed as percentages of inhibition with drugs and standard deviations from means of at least three separate experiments, each performed in quadruplicate.

3.3. Effect of drug combination Combining bafilomycin A1 (1 ␮M) and vanadate (100 ␮M) significantly reduced the vacuolar ATPase activity of D10 by 70.0 ± 4.8% and RSA11 by 73.7 ± 7.3% when compared to their individual effects (Fig. 1), which could indicate different sites of inhibition for the proton pump and Pgp inhibitors. However, various concentrations of bafilomycin A1 and vanadate should be tested individually to determine whether the concentrations used in combination already led to a plateau. Thus, it cannot be excluded that these drugs acted on the same target. 3.4. Effect of chemosensitisers Verapamil (10 ␮M), trifluoperazine (10 ␮M) and progesterone (100 ␮M) had no effect on the vacuolar ATPase activities of the strains tested (Table 3). Of the chemosensitisers tested, trifluoperazine (10 ␮M) and progesterone (100 ␮M) inhibited D10 plasma membrane ATPase activities by 29 ± 13% and 31 ± 24%, respectively. 3.5. Effect of antimalarials Reduced ATPase activity of P. falciparum was reported using 1 mM chloroquine, 1 mM mefloquine and 0.1 mM quinine [31]. The concentrations of the antimalarials tested in this study were based on those shown by Choi and Mego [31] to reduce the Plasmodium food vacuole ATPase activity. The weak base antimalarials mefloquine (1 mM), quinine (0.1 mM) and amodiaquine (0.1 mM) had no significant effect on the vacuolar ATPase activity of any of the strains used (Table 4), suggesting that vacuolar proton pump activity is not stimulated by their sequestration of protons. Mefloquine (1 mM) and quinine (0.1 mM) inhibited the ATPase activity of membranes isolated from the chloroquine-resistant strain Fac8 by 39% and 29%, respectively, while these antimalarials had no effect on the

Detailed sequences and structure analysis of Ca2+ -ATPase proteins have been described [32–36] and the parasite calcium ATPase activity has been localised in both the plasma and parasitophorous membrane [37]. Functional assay of PfATP4 has confirmed that the Ca2+ -ATPase activity observed was mediated by P-type ATPases [38]. In a previous study, we found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes, whereas activity was preserved when Mn2+ was substituted for Mg2+ [25]. In view of these results, the parasite plasma membrane ATPase activity was found to be similar to the vacuolar membrane ATPase activity. Considering that 106 vacuoles correspond approximately to a protein content of 1 ␮g, the ATPase activity of plasma membranes was found to be of approximately within a 10-fold range to that of digestive vacuoles. It should also be noted that protein contents of digestive vacuoles includes soluble intravacuole and membrane proteins whereas plasma membrane protein contents refers only to membrane vesicles. The level of ATPase activity measured in the plasma membranes and the digestive vacuoles did not follow the same pattern (Table 1). Plasma membranes isolated from RSA11 displayed a similar ATPase activity to those isolated from Fac8 while the ATPase activity of RSA11 digestive vacuoles had much lower values than that of Fac8 digestive vacuoles. In addition, no linear relationship was observed between the level of ATPase activity and chloroquine sensitivity. Vacuolar ATPase activity of the chloroquinesensitive D10 and the chloroquine-resistant RSA11 were 65.86 and 68.85 nmol Pi/min/mg protein, respectively. Plasma membranes isolated from the chloroquine-sensitive D10 and RSA3 displayed an ATPase activity of 3.25 and 4.17 nmol Pi/min/mg protein, respectively, whereas those isolated from the chloroquine-resistant Fac8, RSA11 and K1 displayed an ATPase activity of 11.90, 10.05 and 5.81 nmol Pi/min/mg protein, respectively. In this study, the standard error of the ATPase assay was found to vary from 1.4% to 24%. Therefore, changes of less than 25% in ATPase activity were considered insignificant. Chloroquine accumulation in resistant P. falciparum parasites was observed to be more sensitive to the V-type ATPase inhibitor bafilomycin A1 than its sensitive counterparts which lead to the proposal that a weakened vacuolar proton pumping activity could be responsible for reduced chloroquine accumulation [13]. However, this study showed that chloroquinesensitive and -resistant parasites display similar vacuolar ATPase in the presence of bafilomycin A1 and NEM (Table 2) and thus failed to provide evidence for a weakened proton pump activity in purified food vacuoles from chloroquine-

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Table 3 Effect of the chemosensitisers, verapamil (10 ␮M), trifluoperazine (10 ␮M) and progesterone (100 ␮M), on P. falciparum plasma membranes and vacuolar ATPase activity Chemosensitiser

Percentage of inhibitiona Plasma membranes

Verapamil Trifluoperazine Progesterone

Digestive vacuoles

D10

Fac8

D10

Fac8

RSA11

6 ± 14 29 ± 13 21 ± 24

21 ± 17 9 ± 32 12 ± 24

13.6 ± 2.7 0.5 ± 3.4 9 ± 7.9

2.7 ± 5.6 NDb 13.2 ± 7.7

12.7 ± 3.1 7.8 ± 10.7 7.5 ± 3.8

a Values are expressed as percentages of inhibition of ATPase activity compared to controls without drugs and standard deviations from means of at least three separate experiments, each performed in quadruplicate. b ND: not determined.

resistant P. falciparum. In addition, vacuolar ATPase activity did not decrease with decreasing chloroquine sensitivity of a strain (Table 1) as might be predicted by the presence of a weakened vacuolar proton pump in resistant strains. Vacuolar ATPase activity was elevated in the moderately chloroquineresistant Fac8 while D10 and RSA11, which demonstrate an approximate 10-fold difference in chloroquine sensitivity, had similar but lower values. These results together with the lack of correlation between the inhibitory effects of the various inhibitors tested on the ATPase activity of the digestive vacuole and the chloroquine phenotype of the strain appears to indicate that proton pumping activity is not linked to chloroquine resistance. On the other hand, bafilomycin A1 reduced by 30% the ATPase activity of plasma membranes isolated from D10 when compared to those isolated from Fac8, suggesting the presence of a V-type ATPase in D10 plasma membrane that is underexpressed or altered in the plasma membrane of the chloroquine-resistant Fac8. Similarly, NEM, a known inhibitor of the proton pump decreased by 32% and 14%, respectively, the ATPase activity of plasma membranes isolated from D10 and Fac8. Our results are consistent with the reported subcellular distribution of the V-type-H+ -ATPase B-subunit within the parasite, shown to be located in both the plasma and vacuolar membrane [8,15]. The acidic pH of the vacuole is thought to play a critical role in its various functions including the mechanism of chloroquine resistance [3,39]. However, the mechanisms by which the pH within this organelle is maintained are not well understood. It has been suggested that

alteration in the digestive vacuole pH is also responsible for the mechanism of resistance reversal [4,39]. Recently, It has been argued that a V-type ATPase is the major mechanism accounting for the H+ -efflux occurring in both the cytoplasm [16] and the digestive vacuole [40]. Although the genes encoding the A and B subunits of the P. falciparum proton pump have been sequenced, so far, no mutations or overexpression of these genes which could explain chloroquine resistance has been reported [8,14]. Pgh1, a P-type ATPase was identified on the P. falciparum plasma and vacuolar membrane and was believed to play a role in both the mechanism of chloroquine accumulation and chloroquine-resistance [9]. Reduced drug uptake is responsible for resistance, which has been incompletely associated with changes in vacuolar membrane protein Pgh1 [6]. Whether Pgh1 transports chloroquine [2] or controls its uptake through regulation of the intravacuolar pH by a chloride channel activity [41,42] is still subject of controversy. In addition to Pgh1, another vacuolar membrane protein, PfCRT [5] has been proposed to play a role in chloroquine resistance [3,43,44] either as a chloride channel or transporter [45,46]. In this study, the effect of P-type ATPase inhibitors on the ATPase activity of plasma membranes was observed in order to determine their participation in the ATPase activity measured (Table 2). Vanadate reduced both the plasma membrane and vacuolar ATPase activity therefore implicating a P-type component in the ATPase activity observed since the V-ATPases are not sensitive to vanadate [47]. Furthermore, the inhibition of Fac8 ATPase activity by oligomycin both

Table 4 Effect of the common antimalarials, mefloquine (1 mM), quinine (0.1 mM) amodiaquine (0.1 mM) and artemisinin (1 mM), on P. falciparum plasma membranes and vacuolar ATPase activity Antimalarial

Percentage of inhibitiona Plasma membranes

Mefloquine Quinine Amodiaquine Artemisinin

Digestive vacuoles

D10

Fac8

D10

Fac8

RSA11

21 ± 17 16 ± 14 NDb 11 ± 6

39 ± 12 29 ± 20 NDb 4 ± 24

−3.6 ± 10.6 1.1 ± 8.4 3.4 ± 13.4 NDb

−3.8 ± 10.2 −0.3 ± 3.0 −1.4 ± 1.5 NDb

−2.3 ± 4.5 6.2 ± 5.4 7 ± 4.3 NDb

a Values are expressed as percentages of inhibition of ATPase activity compared to controls without drugs and standard deviations from means of at least three separate experiments, each performed in quadruplicate. b ND: not determined.

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in the digestive vacuoles (61%) and the plasma membranes (58%) would be consistent with possible higher levels of Pgh1 in Fac8 [9] since this inhibitor has been shown to inhibit Pgp homologues [48,49]. Many compounds that reverse MDR in cancer cells have been identified [50]. These chemosensitisers were shown to have a stimulatory effect on P-glycoprotein ATPase activity in plasma membranes [51–53], Pgp reconstituted in liposomes [54] and purified P-glycoprotein [28]. Similarly, chloroquine resistance can be reversed by a certain number of chemically distinct compounds [55–61]. It is difficult to predict how chemosensitisers might affect the ATPase activity of the parasite plasma membranes, since the mechanism by which they reverse chloroquine resistance is unknown. Moreover, their mode of action might vary depending the type of chemosensitiser. The three chemosensitisers chosen in this study belong to various classes of compound known to sensitise MDR cells to chemotherapeutic agents in mammalian cancer cells [50]. These include the calcium channel blocker verapamil, the calmodulin inhibitor trifluoperazine and the steroid hormone progesterone. A study conducted in our laboratory [62] determined the dose–response effects of verapamil, trifluoperazine and progesterone on increasing the level of chloroquine accumulation in a chloroquine-resistant strain. The maximum levels of chloroquine accumulation were obtained at 5 and 3 ␮M for verapamil and trifluoperazine, respectively, whereas none of the progesterone concentrations tested potentiated chloroquine accumulation. In this study, of the chemosensitisers tested, trifluoperazine (10 ␮M) and progesterone (100 ␮M) inhibited D10 plasma membrane ATPase activities by 29% and 31%, respectively and none of these chemosensitisers had an effect on the ATPase activity of the food vacuole (Table 3). The effect observed in the presence of trifluoroperazine and progesterone is likely to result form the inhibition of an ATPase activity that is not related to a mechanism of chloroquine transport since only the activity of plasma membranes isolated from the chloroquine-sensitive strain were affected. If the chemosensitisers act directly on an efflux pump, it would indicate that this activity has not been stimulated. However, the target of these chemosensitisers has not yet been identified in P. falciparum and these results would suggest either that the target of chemosensitiser is absent from the parasite plasma membrane and the isolated digestive vacuole or their mode of action does not involve an ATP-dependent mechanism. Nevertheless, it would be interesting to test the effect of these chemosensitisers on the ATPase activity over a range of concentrations, since it has been shown that drugs like trifluoperazine or verapamil have a biphasic effect on Pglycoprotein ATPase activity, producing a stimulation of activity a low concentrations, followed by inhibition at higher concentrations [28,63]. Mefloquine, quinine and artemisinin were reported to inhibit the vacuolar ATPase activity [31]. In contrast, we found that mefloquine and quinine had no effect on the vacuolar ATPase activity whereas an inhibitory effect (39%) was observed on the ATPase activity of plasma membranes

isolated from the chloroquine-resistant strain Fac8 (Table 4). These results could indicate that an ATP-driven drug transport does not occur in the digestive vacuoles but might be involved at the plasma membrane level. However, this study should be extended to a greater number of strains of P. falciparum to determine whether these antimalarials could inhibit the ATPase activity of plasma membranes. Purified digestive vacuoles have been demonstrated to accumulate chloroquine to a much lesser extent than their counterpart infected-erythrocytes [26] suggesting a possible role for the plasma membranes in accumulating chloroquine. However, another likely explanation could be that these isolated vacuoles do not produce heme therefore impeding drug accumulation as the result from a lack of target binding. Although purified digestive vacuoles can be used as a model to characterise vacuolar functions, their value might be limited for the investigation of quinoline transport. These data would therefore be consistent with the hypothesis that chloroquine and related antimalarials act by complexing ferriprotoporphyrin IX (Fe(III)PPIX), inhibiting its conversion to beta-hematin and hence its detoxification [64,65]. Therefore, the saturable accumulation of chloroquine is believed to result from intracellular binding to ferriprotoporphyrin IX rather than active transport into the parasite [66]. Recently, artemisinin has been shown to inhibit the SERCA orthologue (PfATP6) of P. falciparum in xenopus oocytes and this effect was abrogated by chelation of iron with desferrioxamine, which lead to the proposal that artemisin acts by inhibiting PfATP6 after activation by iron [67]. The lack of plasma membrane ATPase inhibition by artemisinin observed in this study would be consistent with this hypothesis. In conclusion, we have characterised the ATPase activity of purified digestive vacuoles and plasma membranes from P. falciparum strains and found no link between proton pumping activity and chloroquine resistance. On the other hand, these results support the view of an ATPase activity associated with a V-type ATPase in the plasma membranes and digestive vacuoles isolated from the chloroquine-sensitive strain of P. falciparum. However, it would be interesting to extend this study to a greater number of P. falciparum strains to determine any correlations between ATPase activities and either the drug uptake capabilities or drug sensitivity of a strain.

Acknowledgement This work was supported by the South African Medical Research Council.

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