©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Enhanced Transport of L- and D-Lactate into Human Red Blood Cells Infected with Plasmodium falciparum Suggests the Presence of a Novel Saturable Lactate Proton Cotransporter (*)

Susan L. Cranmer (§) , Alan R. Conant , Winston E. Gutteridge (1), Andrew P. Halestrap (¶)

From the (1)Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD and The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human erythrocytes parasitized with the malarial protozoan Plasmodium falciparum showed rates of L-lactate, D-lactate, and pyruvate uptake many fold greater than control cells. Thus it was necessary to work at 0 °C to resolve true initial rates of transport. Studies on the dependence of the rate of transport on substrate concentration implied the presence in parasitized cells of both a saturable mechanism blocked by -cyano-4-hydroxycinnamate (CHC) and a nonsaturable mechanism insensitive to CHC. The former was dominant at physiological substrate concentrations with K values for pyruvate and D-lactate of 2.3 and 5.2 mM, respectively, with no stereoselectivity for L- over D-lactate. CHC was significantly less effective as an inhibitor of lactate transport in parasitized erythrocytes than in uninfected cells, whereas p-chloromercuribenzenesulfonate, a potent inhibitor in control cells, gave little or no inhibition of lactate transport into parasitized erythrocytes. Inhibition of transport into infected cells was also observed with phloretin, furosemide, niflumic acid, stilbenedisulfonate derivatives, and 5-nitro-2-(3-phenylpropylamino)benzoic acid at concentrations similar to those that inhibit the lactate carrier of control erythrocytes. These compounds were more effective inhibitors of the rapid transport of chloride into infected cells than of lactate transport, whereas CHC was more effective against lactate transport. This implies that different pathways are involved in the parasite-induced transport pathways for lactate and chloride. The transport of L-lactate into infected erythrocytes was also inhibited by D-lactate, pyruvate, 2-oxobutyrate, and 2-hydroxybutyrate. The intracellular accumulation of L-lactate at equilibrium was dependent on the transmembrane pH gradient, suggesting a protogenic transport mechanism. Our data are consistent with lactate and pyruvate having direct access to the malarial parasite, perhaps via the proposed parasitophorous duct or some close contact between the host cell and parasite plasma membranes, with transport across the latter by both a proton-linked carrier (CHC-sensitive, saturable, and the major route) and free diffusion of the undissociated acid (CHC-insensitive, unsaturable, and a minor route).


INTRODUCTION

Once inside the red blood cell, the malarial parasite Plasmodium falciparum reproduces asexually, producing about 16 progeny every 48 h. Such rates of multiplication require intense metabolic activity, in stark contrast to the meager metabolic requirements of the uninfected erythrocyte. In particular, the energy needs of the parasite, which is a homolactate fermenter, are accompanied by a 20-100-fold increase in the rate of glucose uptake and lactic acid extrusion by the infected cells(1, 2, 3, 4, 5) . Since the parasite is surrounded by three membranes, its own plasma membrane, the parasitophorous vacuole membrane and the host cell plasma membrane, the lactic acid must be transported across all three of them. Alternatively, a parasitophorous duct might be present which would allow direct access of the parasite to the extracellular medium(6) , in which case only a transport mechanism across the parasite plasma membrane would be required. Since glycolysis by the parasite will produce lactic acid, this is most likely to be a lactate/proton cotransporter as is now well established for the lactate transporter of mammalian cells(7) .

If the parasitophorous duct does not exist, a new pathway for lactic acid transport out of the host cell would need to be induced in the host cell membrane since the activity of the endogenous transporter is insufficient to account for the increased fluxes of lactic acid observed in infected cells(4, 7) . Studies from many laboratories have demonstrated that the permeability properties of the host red blood cell plasma membrane are substantially altered by the trophozoite stage of infection. Thus, rates of entry of amino acids, glucose, nucleosides, choline, anions, and cations into the infected red cell are all increased, as is the permeability to other compounds, such as mannitol, which are normally impermeant(5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . It is probable that the enhanced permeability is caused by the export of parasite-derived proteins to the host plasma membrane, but parasite-induced changes in the properties of host cell plasma membrane proteins such as Band 3 cannot be ruled out(19, 20, 21, 22, 23, 24) . It is also unclear whether there are a variety of new transport pathways induced in the infected cell or whether a nonspecific channel is opened which allows entry of any small molecule, but with a preference for anions(18) . The enhanced entry of many different compounds into infected cells is unsaturable and inhibited in a similar manner by a range of inhibitors, many of which are chloride channel blockers(4, 5, 13, 17, 18) . However, in the case of nucleosides and choline, evidence has been presented for the presence of two transport pathways in parasitized cells, one saturable and one not(12, 15, 16, 25) . A complication of studying substrate transport into infected cells is that it is difficult to discriminate between substrate uptake into the cytosol of the red cell and uptake into the parasite itself. It is possible that the saturable and unsaturable components of transport which have been described represent these two processes.

In the present paper we provide a detailed account of the characteristics of lactate and other monocarboxylates transported into human red cells infected with P. falciparum. We show the presence of both a saturable and an unsaturable proton-linked transport mechanism in parasitized cells. Our data are most easily interpreted if lactate and pyruvate have direct access to the malarial parasite, perhaps via the proposed parasitophorous duct or some close contact between the host cell and parasite plasma membranes, with transport across the latter occurring by both a proton-linked carrier (CHC()-sensitive, saturable, and the major route) and free diffusion of the undissociated acid (CHC-insensitive, unsaturable and a minor route).


EXPERIMENTAL PROCEDURES

Materials

L-[C]Lactate, D-[C]lactate, [1-C]pyruvate, HO, and NaCl were obtained from Amersham International, Amersham, Bucks., U. K. [1-C]Pyruvate was dissolved in water and divided into 2.5-µCi aliquots that were freeze-dried and stored at -20 °C. Stilbenedisulfonate derivatives were obtained as described previously(26, 27) . 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was obtained from Research Biochemicals Inc., Natick, MA. Silicone oil MS550 and dinonyl phthalate were obtained from BDH Chemicals, Poole, Dorset, U. K. Unless stated otherwise, all other chemicals and biochemicals were obtained from the sources given previously(28, 29) . Acetazolamide was used as a 0.1 M stock solution in dimethyl sulfoxide and stored at -20 °C. 4,4-Diisothiocyanostilbene-2,2`-disulfonate (DIDS) was made up freshly prior to use as a 2.5 mM solution in HO.

Recently donated A erythrocytes and human serum, types A, AB, or AB, were obtained from the South West Regional Blood Transfusion Centre, Southmead Hospital, Bristol, U. K. P. falciparum, strain ITO4, used for all experiments, was a gift of Dr. Barry Elford, University of Oxford, U. K.

Culture of Malaria-infected Erythrocytes

P. falciparum, strain ITO4, was grown under continuous culture conditions following the method of Trager and Jensen(30) . Infected A erythrocytes were cultured in RPMI 1640 medium containing 20 mM HEPES (Life Technologies, Inc. Ltd., Paisley, U. K.), supplemented with 5.5 mM glucose (BDH), 2 mM glutamine (Sigma, Poole, U. K.), hypoxanthine 12.5 mg/liter (Life Technologies, Inc.), gentamicin sulfate (Life Technologies, Inc.) at 10 mg/liter and 10% human serum, types A, AB, or AB. Infected cells were synchronized by either gelatin flotation or sorbitol hemolysis and harvested, at the trophozoite stage, by gelatin flotation(31, 32, 33) . Parasitemia was estimated from methanol-fixed Giemsa-stained smears.

Radioactive Influx Measurements

We have used the silicone oil centrifuge stop technique (7, 34) to measure the uptake of lactate and pyruvate into control and parasitized erythrocytes. In all experiments the substrate was labeled with C, and HO was also added to allow for correction of total water (intracellular and extracellular water) in the pellet. Parallel experiments were always performed to measure the intracellular volume using [C]sucrose and HO, and these values were used in conjunction with the total cell water in the uptake experiment to convert total C present in the pellet to intracellular C (7, 34). Measurements of intracellular pH were also made routinely using 0.5 mM [2-C]dimethyloxazolidine-2,4-dione (DMO) (35) since lactate transport is usually proton-linked and is driven by the pH gradient across the plasma membrane(7) . Transport can thus be maximized by generating an alkaline intracellular pH, and this was achieved in the present experiments through the use of a citrate buffer (7, 36).

Prior to measurement of transport, both infected and uninfected cells were depleted of intracellular lactate by two sequential incubations in bicarbonate-buffered saline for 30 min at 37 °C, as described previously(29) , before resuspending in citrate buffer (78 mM sodium citrate, 15 mM HEPES, 1 mM EGTA, adjusted to pH 7.4 with NaHPO4), containing 100 µM acetazolamide. Cells were then incubated with DIDS (5 µM) at 0 °C for 1 h to ensure complete inhibition of lactate transport via the anion transporter, Band 3(36) . Uptake of C-substrate was initiated by rapidly mixing 100 µl of erythrocytes (5% hematocrit) with 10 µl of citrate buffer containing C-radiolabeled substrate (1 µCi/ml), HO (10 µCi/ml), and unlabeled substrate at the required concentration. The resulting 110-µl sample was transferred to a 0.3-ml microcentrifuge tube containing 100 µl of oil (specific gravity 1.035) (MS550 silicone oil:dinonyl phthalate; 65:35 v/v) layered on top of 50 µl of 10% (v/v) HClO containing 25% (v/v) glycerol. The cells were carefully placed just above the oil layer and uptake terminated at the required time by centrifugation at 10,000 g for 30 s in a Beckman Microfuge E. The cells sedimented through the oil layer into the HClO, and the tubes were subsequently frozen in liquid N. The tip containing the HClO/glycerol layer and cell pellet was cut off, placed in a scintillation vial, and mixed with 800 µl of HO until the pellet had thawed. After the addition of 10 ml of scintillant (Packard Emulsifier Safe) the vials were capped and vigorously vortex-mixed twice to ensure that all radioactivity was released from the tube. A 20-µl sample of the supernatant was treated in the same manner and radioactivity determined by dual isotope scintillation counting.

For measurement of pH gradients and intracellular volumes the protocol was identical except that C-substrate was replaced by 0.5 mM [2-C]DMO or [C]sucrose (1 µCi/ml), respectively. Intracellular C was calculated as (C-substrate-HO) space - ([C]sucrose-HO) space as described previously(37) .

Presentation of Results

The data for infected erythrocytes were fitted by nonlinear least squares regression analysis to first-order rate Equation 1

On-line formulae not verified for accuracy

where L is the lactate uptake at time t, L is the total uptake of lactate at equilibrium, k is the first-order rate constant, and x is the dead time of centrifugation (the time taken to sediment the cells through the oil layer into perchloric acid), which averaged 8 s. The additional term p represents a component of transport into infected cells which was linear with respect to time and may represent uptake into a different intracellular compartment such as the host cell cytosol or contaminating uninfected erythrocytes. The initial rapid rate of substrate uptake behaved as if only a single intracellular compartment (referred to simply as ``parasitized cells'') was involved, and initial rates of transport were either calculated from k and L. (i.e.k L) or from the uptake at 10 or 15 s assuming uptake to be linear with respect to time over this period (see below).

In most cases data are presented as the means ± S.E. of several separate experiments performed on separate cell cultures. Statistical analysis of differences between data groups was performed using either a paired or unpaired Student's t test as appropriate.


RESULTS

Time Courses of Lactate and Pyruvate Entry into Control and Parasitized Erythrocytes

In Fig. 1we show that when L-lactate uptake was measured at 25 °C using the silicone oil centrifuge stop technique, the time course obtained was similar to that measured previously for human erythrocytes using centrifugation stop in the absence of silicone oil (36). However, for parasitized cells the rate of uptake was greatly enhanced, to the extent that resolution of initial rates was not possible. Similar observations have been made by others(4, 18) . The fastest practical time point for centrifugation stop was 10 s after the addition of C-substrate. This represents a total uptake time of about 18 s when the dead time is included. Other techniques that might allow faster time resolution such as membrane filtration or inhibitor stop (7, 34) are not appropriate for use with parasitized cells. No suitable inhibitor was available for the former technique, whereas for membrane filtration it is necessary to wash the extracellular medium away from the cells on the filter, and this was not possible without losing intracellular substrate(7) . Thus, to resolve the initial rates of transport we decreased the temperature for the uptake experiments to 0 °C. In Fig. 2data are shown for the time courses of L-lactate, pyruvate, and D-lactate transport into control and parasitized cells at 0 °C. These data do allow initial rates of transport to be determined and confirm the great enhancement of transport rates seen in the parasitized cells when compared with control cells. The effect is most noticeable for D-lactate, which is transported little if at all by the normal red cell transporter(7, 38) , and least for pyruvate, which is the best substrate for the native transporter(7, 36, 38) . The lack of stereoselectivity of the uptake into parasitized cells is confirmed in Fig. 2d, where parallel time courses of L- and D-lactate into parasitized cells were determined in the same experiment. The rates of transport of the two isomers was indistinguishable. These data also confirm that the dead time for centrifuge stop is about 8 s and show that the initial rate of transport can be considered linear for at least the first 15-30 s. In subsequent studies, depending on the substrate and its concentration, uptake at 10, 15, or 30 s was used as an estimate of initial rate. It seems likely that previous studies of lactate transport into parasitized erythrocytes which utilized higher temperatures (18) or less accurate time resolution (4) were not capable of resolving initial rates of transport and probably underestimated the true transport rate. The Dependence of Lactate Transport on the pH Gradient-The lactate transporter of uninfected erytrocytes, and indeed most other mammalian cells, is a lactate proton cotransporter (7) which suits the physiological setting in which metabolism both produces and uses lactic acid(7) . Since the enhanced lactate transport across the plasma membrane of the parasitized erythrocyte is required to remove glycolytically derived lactic acid from the cell, it would seem highly probable that the induced transport mechanism(s) were also proton-linked. To test this possibility we have compared the uptake of L-lactate into parasitized cells in both citrate medium (pH gradient 0.75 unit of alkaline inside) with those in a HEPES-buffered saline medium (pH gradient 0.02 alkaline inside). The data are presented in Fig. 3. Although the initial rates of transport were similar in both media (0.58 ± 0.020 and 0.48 ± 0.029 nmol/µl of intracellular space/min in citrate and saline media, respectively), the uptake at equilibrium was enhanced severalfold in the citrate medium compared with the saline medium. The uptake at equilibrium for a proton-linked lactate transporter is given by Equation 2.

On-line formulae not verified for accuracy

Thus, the pH gradient determines the maximum uptake of lactate at equilibrium. In our experiments we routinely measured the pH gradient by replacing the 0.5 mM lactate by 0.5 mM DMO, which should perturb the pH gradient to the same extent as lactate. The pH gradient varied considerably from one experiment to the next but was generally lower when parasitemia was high. However, the correlation was not consistent, and variation in control cells was also considerable from day to day. This inherent variation, and the use of saline media in which little pH gradient is maintained, allowed us to study the relationship between the intracellular concentration of lactate after equilibrium had been reached with the pH gradient in both control and parasitized cells. The data are presented in Fig. 4. It is clear that in both cases the data fall on a similar line, strongly indicative of a protogenic mechanism.


Figure 1: Time courses for the uptake of L-lactate into parasitized and uninfected erythrocytes at 25 °C. The uptake of 0.5 mML-lactate into parasitized () and control () erythrocytes was measured as described under ``Experimental Procedures.'' Data are presented as means ± S.E. (error bars) of three separate experiments and were fitted to a first-order rate equation. The pH values (mean ± S.E.) for parasitized and control erythrocytes were 0.53 ± 0.1 and 0.97 ± 0.14, respectively, and the parasitemia ranged between 39.4 and 78.2%.




Figure 2: Time courses for the uptake of pyruvate and L- and D-lactate into parasitized and uninfected erythrocytes at 0 °C. The uptake of 0.5 mML-lactate (panels a and d), pyruvate (panel b), and D-lactate (panels c and d) into parasitized () and control () erythrocytes was measured as described under ``Experimental Procedures.'' Data are presented as the means ± S.E. (error bars) of three separate experiments and were fitted to a first-order rate equation. The pH values (mean ± S.E.) for parasitized and control erythrocytes were 0.72 ± 0.05 and 1.01 ± 0.06, respectively, and the parasitemia ranged between 40 and 90%. In panels a-c different preparations of parasitized cells were used for each substrate, whereas in panel dL-lactate (, dashed line) and D-lactate (, solid line) uptake were measured in paired experiments on the same preparations of parasitized cells.




Figure 3: Comparison of the time courses of L-lactate uptake into parasitized erythrocytes in citrate or HEPES-buffered saline medium. The uptake of 0.5 mML-lactate into parasitized erythrocytes was measured at 0 °C in citrate medium () or HEPES-buffered saline medium (). Data are presented as means ± S.E. (error bars) of three separate experiments and were fitted to a first-order rate equation with a dead time set at 8 s as described under ``Experimental Procedures.'' The initial rates (± S.E. of the fit shown) were 0.58 ± 0.02 and 0.48 ± 0.03 nmol/µl of intracellular space/min for cells incubated in citrate and saline media, respectively. The corresponding pH values (means ± S.E.) were 0.75 ± 0.05 and 0.02 ± 0.01, respectively, and the parasitemia ranged between 70 and 80%.




Figure 4: The relationship between the equilibrium uptake of L-lactate and the pH gradient for parasitized and uninfected erythrocytes. Data for equilibrium uptake of L-lactate (after 5 min) into parasitized erythrocytes at 0 °C (,) or control erythrocytes at 25 °C (,) were taken from separate experiments performed in citrate medium (,) or HEPES-buffered saline medium (,). The line drawn is a linear regression of the data (r = 0.939, p = 1.27 10).



The Concentration Dependence of D-Lactate and Pyruvate Transport

The concentration dependence of the initial rates of both pyruvate and D-lactate transport were determined, and the data are presented in Fig. 5. For this purpose the uptake of substrate at 10 s (pyruvate) or 15 s (D-lactate) was taken to represent initial rates of uptake, and this was confirmed at both the highest and lowest substrate concentration used. D-Lactate was used in preference to L-lactate in these experiments to avoid any possibility that uptake into contaminating nonparasitized cells might interfere. However, from the data of Fig. 2d it is clear that this effect is likely to be small; in a single experiment in which the concentration dependence of L- and D-lactate transport was studied in parallel, the results were indistinguishable (not shown). It should be noted that the uptake of substrate at these short times was not great, and by necessity the resulting error on replicate determinations was of the order of 20%. Furthermore, the variable parasitemia in the parasitized erythrocyte preparation also introduced some variation. For these reasons the results of several experiments were meaned before determination of the kinetic parameters of transport. For both D-lactate and pyruvate the initial rate of transport was clearly not linear with respect to substrate concentration at lower concentrations, but a linear relationship was observed at higher concentrations. The data were best fitted to an equation incorporating both a saturable component and a nonsaturable component as described previously(37) . In the presence of 2 mM CHC, only the unsaturable component remained, suggesting that this may represent either diffusion of the free undissociated acid across the membrane or the presence of an anionic channel as observed in both isolated heart and liver cells, especially at low temperatures(7, 27, 35, 36) . The K values (± S.E. for the fit shown) for pyruvate and D-lactate were 2.25 ± 0.45 and 5.20 ± 0.82 mM, respectively, whereas the corresponding V value (at 0 °C) for pyruvate was 3.84 ± 0.57 nmol/min/µl of intracellular space and was fixed at the same value for D-lactate. These values compare with K values for pyruvate, L-lactate, and D-lactate of 1.9, 9.1, and >50 mM in uninfected cells with a corresponding V value (at 10 °C) of of 2.0 nmol/min/µl of intracellular space. The V of the lactate transporter of uninfected cells at 0 °C can be calculated from the activation energy for transport between 0 and 10 °C of 148 kJmol to be 0.2 nmol/min/µl of intracellular space. Thus, the maximal rate of carrier-mediated transport is increased about 20-fold in parasitized cells.


Figure 5: Concentration dependence of the initial rate of pyruvate and D-lactate uptake into parasitized erythrocytes. The uptake of [C]pyruvate (panel a) or D-lactate (panel b) was measured after a 10- and 15-s incubation at 0 °C, respectively, over which period uptake was linear with time (Fig. 2). Data are presented as the means ± S.E. (error bars) of five (pyruvate) or three (D-lactate) experiments in the absence (solid lines and closed symbols) or presence (dashed lines and open symbols) of 2 mM CHC. The initial rates of transport were calculated assuming a dead time of 8 s. The data in the absence of CHC were fitted to the equation describing the kinetics of a mechanism combining a saturable and unsaturable component: v = V[S]/(K + [S]) + k[S], where v is the initial rate of transport at substrate concentration [S], V is the maximal velocity of the carrier, K is the Michaelis constant, and k is the first-order rate constant for the nonsaturable component. For pyruvate all parameters were fitted by least squares regression analysis and yielded values for V, K, and k (± S.E.) of 3.84 ± 0.57 nmol/min/ml of intracellular space, 2.25 ± 0.45 mM, and 0.45 ± 0.03 min, respectively. For D-lactate the V was fixed at the same value and the values for K and k determined as 5.20 ± 0.82 mM and 0.64 ± 0.02 min, respectively. Data in the presence of 2 mM CHC were fitted by linear regression to give slopes of 0.616 ± 0.014 and 0.417 ± 0.023 min for pyruvate and D-lactate, respectively.



Substrate Specificity of the Induced Lactate Transporter of Parasitized Erythrocytes

To investigate the substrate specificity of the induced transport pathway we have studied the ability of other monocarboxylates to inhibit lactate transport into parasitized erythrocytes. We have previously used this technique successfully in studies of the substrate specificity of the lactate transporter of control erythrocytes and cardiac myocytes(7, 29, 36) . The data are presented in , where results are also presented for control erythrocytes incubated under identical conditions. The inhibition given by D-lactate and pyruvate (32.6 and 43.5%, respectively) are close to the values of 30 and 50%, respectively, which would be predicted for these substrates acting as competitive inhibitors if it is assumed that the K and K values are the same. This assumption is valid for the lactate carrier of other cells(7) . The data of also demonstrate that 2-oxobutyrate and 2-hydroxyisobutyrate are substrates for the transporter of parasitized cells, whereas only the former is a substrate of the carrier of control cells as shown previously(7) .

Inhibitor Specificity of Lactate Transport into Parasitized Erythrocytes

Work from this and other laboratories has characterized a wide range inhibitors of the lactate transporter of erythrocytes (see 7). These include the cyanocinnamates such as CHC, stilbenedisulfonates such as DIDS, organomercurials such as p-chloromercuribenzenesulfonate (PCMBS), and miscellaneous compounds such as phloretin and niflumic acid. In the effects of these inhibitors on the initial rate of L-lactate uptake into parasitized erythrocytes at 0 °C are compared with their inhibition of transport into control erythrocytes at 25 °C. In the majority of cases the extent of inhibition was similar, but there were some significant differences. Thus PCMBS, which is a very potent inhibitor of lactate transport into control cells(38) , was almost without effect on transport into parasitized cells. CHC was significantly less effective in the parasitized cells than control cells, but it should be noted that the K of CHC for monocarboxylate transport is critically dependent on both the transmembrane pH gradient and the temperature(7, 39) . Although the inhibition (mean ± S.E.) by 2 mM CHC of the rate of transport of 0.5 mML-lactate and D-lactate was 86.1 ± 5.8% (10) and 79.8 ± 9.4% (n = 3), respectively, that for 0.5 mM pyruvate was only 67.0 ± 4.1%(3) . At 5 mMD-lactate or pyruvate the inhibition dropped to only 62.5 ± 5.4% (3) and 41.9 ± 7.0%(3) , respectively (see also Fig. 5). These results are consistent with CHC inhibiting only the carrier-mediated monocarboxylate transport. Since the rate of the CHC-insensitive transport increases linearily with substrate concentration (Fig. 5) this pathway becomes proportionately greater at higher substrate concentrations. In contrast to CHC and PCMBS, furosemide, and NPPB were substantially more effective at inhibiting transport into parasitized cells than control cells. Nonetheless, it should be noted that NPPB is still a very potent inhibitor of the monocarboxylate carrier in control erythrocytes, the K value, determined using variable concentrations of NPPB at 0.5 mML-lactate as described previously(26) , being 8.5 ± 0.9 µM (mean ± S.E. of five separate experiments).

We have confirmed that the same pattern of inhibition observed when L-lactate was used as substrate was also observed when D-lactate was used. These data are also presented in . We also investigated the effects of 40 µM phloridzin on the initial rate of transport of D-lactate transport into the infected erythrocytes and found 59.5 ± 4.0% inhibition. This concentration of inhibitor had no effect on L-lactate transport into control erythrocytes.

Comparison of the Relative Potency of Inhibitors against Chloride and Lactate Transport into Parasitized Erythrocytes

Kirk et al.(18) have suggested that the increase in permeability of parasitized erythrocytes to a wide range of compounds, including lactate, is the result of the induction of an anion-selective channel in the erythrocyte plasma membrane. The main evidence for this was that entry of a wide range of compounds including lactate and chloride was inhibited with similar potency by well established anion channel inhibitors. To test whether the enhanced rate of entry of lactate and pyruvate could be accounted for by such a mechanism we compared the inhibition by a range of compounds of the initial rate of 0.6 mML-lactate and Cl in paired experiments. The results are presented in I. In these experiments Cl (and lactate) transport into uninfected cells mediated by Band 3 was totally inhibited by the addition of 5 µM DIDS. The infected cells showed greatly enhanced rates of Cl entry in agreement with Kirk et al.(18) . Indeed, from time courses of Cl uptake at 0 °C (data not shown) it was found to be necessary to measure transport at 10 s to ensure that true values for initial rates of transport were determined. The rate of transport of 0.6 mM Cl measured in this way was 1.31 ± 0.03 nmol/µl/min (mean ± S.E. of five observations) compared with a value of 0.84 ± 0.06 for L-lactate transport. The initial rate of Cl transport was strongly inhibited by 2.5 µM NPPB, 25 µM niflumate, 200 µM DIDS, and 25 µM furosemide. Surprisingly, 2.5 mM pyruvate and 5 mML-lactate also inhibited the initial rate of Cl uptake by 27.4 ± 6.6 and 39.2 ± 8.7%, respectively (I). In contrast Cl does not appear to inhibit the rate of lactate transport since the initial rates observed in citrate medium (no Cl) and saline medium (150 mM Cl) were very similar (0.58 ± 0.020 and 0.48 ± 0.029 nmol/µl intracellular space/min, respectively; Fig. 3). When the effects of inhibitors were tested on the initial rates of lactate and Cl transport in paired experiments (I) it was found that for 200 µM DIDS and 25 µM furosemide, the inhibition of both processes was of a similar magnitude. For 2.5 µM NPPB and 25 µM niflumate the inhibition of lactate transport was significantly less than for Cl, whereas 200 µM CHC gave sigificantly greater inhibition of lactate transport than that of Cl. At 2 mM CHC inhibition of L-lactate and Cl transport was 88.3 ± 5.0 and 96.6 ± 4.7%, respectively, whereas for 100 µM NPPB, which totally inhibited chloride transport, the inhibition of D-lactate transport was 83.4 ± 4.3%. Thus, the residual lactate transport that occurs in the presence of CHC or NPPB is unlikely to be due to the induced anion channel proposed by Kirk et al.(18) . Taken together, these data imply that lactate and Cl do not enter the parasitized cell by an identical mechanism, although they do not rule out some lactate entering by the same mechanism as Cl.


DISCUSSION

The data we present in this paper confirm and greatly extend the observations of Kanaani and Ginsburg(4) , who also demonstrated a dramatic increase in L-lactate transport into malaria-infected cells which was inhibited by CHC but not by PCMBS. These authors were unable to demonstrate saturation of the transport mechanism, but their time resolution for transport measurements was not sufficient to allow determination of true initial rates. In contrast, our measurements at low temperature did allow us to determine initial rates of transport and demonstrated that there is a saturable component of the transport mechanism. Our data ( Fig. 3and Fig. 4) imply that this new lactate transport mechanism is protogenic like that found in most other cells(7) . This is what might be predicted, since the parasite's glycolytic pathway produces lactic acid rather than lactate. Indeed, the V of the transporter is similar to that determined for Ehrlich Lettre tumor cells, which are also almost exclusively glycolytic for their energy requirements(39) .

In addition to the saturable component there also appears to be a nonsaturable component, not blocked by CHC, which becomes dominant at higher substrate concentrations. This may represent either free diffusion of the undissociated acid or transport via an anionic channel as has been suggested in other cells(7, 35, 37) . Saturable and nonsaturable components of the induced transport mechanism for choline and nucleosides into parasitized erythrocytes have also been described by some (12, 15, 16, 25) but not all workers(13) . If an anionic pathway is responsible for the nonsaturable component of lactate transport, it is unlikely to be that described by Kirk et al.(18) since it is blocked by neither CHC nor NPPB at concentrations that almost totally inhibit Cl transport into infected cells. This conclusion is strengthened by the demonstration that there are inhibitors such as CHC which inhibit L-lactate transport into the parasitized cells more than Cl, whereas others, such as NPPB and niflumate, are less potent toward lactate than Cl.

A question that our data cannot resolve with certainty is the location of the new transport pathway(s). For lactic acid to enter or leave the parasite there are potentially three membranes that must be crossed: the host cell plasma membrane, the parasitophorous vacuolar membrane, and the parasite plasma membrane. Transport across all three would have to be very rapid to keep up with the parasite's high rates of glycolysis. However, an alternative possibility has been suggested, which proposes that there is a parasitophorous duct that allows the parasite direct access to the extracellular medium, without the requirement for passage through the host cell cytosol(6) . This proposal is still controversial(40, 41, 42, 43, 44, 45, 46, 47) , but evidence in favor of this proposal has come from several sources. First, confocal microscopy has shown the presence of such a duct(6, 45, 46) , although this is not accepted by all workers(41, 43) . Second, the ability of certain iron chelators and phloridzin to inhibit parasite growth when added directly to the extracellular medium, but not when incorporated into the host cell cytosol by encapsulation, can best be explained by their access to the parasite being direct rather than through the host cell(48, 49, 50) . Third, antibodies added to the outside of infected cells appear to have access to antigens located on the parasitophorous vacuolar membrane (47). Fourth, 2-deoxyglucose can enter the parasite even when the host cell glucose transporter and the induced pore are both blocked by the presence of 50 µM cytochalasin B and 500 µM niflumic acid(51) . Our own data are also most easily explained if there is direct access of lactate to the parasite which it then enters via a CHC-inhibitable proton-linked transporter (saturable) and CHC-insensitive diffusion of the free acid (nonsaturable), both of which would be sensitive to the transmembrane pH gradient. In the absence of the duct, lactate would have to cross the host cell plasma membrane by means of the induced permeability channel described above, followed by passage across the parasitophorous vacuole membrane by means of the large nonspecific channels present in this membrane(52) . Such a process would probably lead to rapid equilibration of lactate with the host cell cytosol before equilibration with the parasite cytosol, but we have found no evidence for more than one compartment transporting lactate at a rate sufficient to account for the observed rates of lactate efflux during active metabolism. However, if the membranes of the host cell, parasite, and parasitophorous vacuole are in close proximity this might effectively allow direct access of lactate to and from the parasite without equilibration with the host cytosol.

Whatever the pathway that lactate takes to and from the parasite plasma membrane, it would seem most likely that a protogenic carrier catalyzes the net efflux of lactic acid out of the parasite as in the case in other highly glycolytic cells(7, 39) . Indeed, in another blood-borne protozoa, Trypanosoma brucei, there is strong evidence for a specific proton-linked pyruvate transporter in the plasma membrane with a K for pyruvate of about 2 mM(53) , similar to the value reported here for the malarial protozoa. The trypanosomal carrier is also inhibited by cyanocinnamate derivatives and DIDS with a potency similar to that observed P. falciparum(54) . However, the trypanosomal carrier appears to be specific for pyruvate and other keto acids and is not inhibited by either L- or D-lactate. This is entirely consistent with the metabolism of T. brucei, which produces and excretes pyruvic acid as an end product of glycolysis. Substantial differences in the affinity of different isoforms of the mammalian lactate carrier for pyruvate and L- and D-lactate are well documented(7) , and thus the trypanosomal and malarial monocarboxylate carriers may be related.

The number of people at risk from malaria worldwide is more than 2 billion, with about 270 million new cases of the disease being reported each year. Of these some 2 million a year will die, mainly from P. falciparum, which is the most severe form of infection. These numbers are set to increase as resistance to the most widely prescribed drugs such as chloroquine becomes widespread(55, 56) . The most important alternative anti-malarial strategy is the development of vaccines. This has proved to be a more complex task than first thought, although prospects remain hopeful(57) . However, new approaches to chemotherapy are still required urgently(58) , and any biochemical feature of the parasite or the infected cell which distinguishes it from a normal red cell is a potential target of drug therapy. Whatever the nature and locus of the induced lactate transport pathway are, the pathway could be a locus for future chemotherapeutic intervention since its inhibition might cause intracellular lactic acid accumulation and consequent impairment of parasite growth. Indeed, Kanaani and Ginsburg (59) have shown that cyanocinnamate derivatives do inhibit malarial parasite growth in culture, as do a variety of other anion transport inhibitors such as phloretin, niflumic acid, and phloridzin(60) , which we have shown also inhibit lactate transport in parasitized cells. However, since these compounds also inhibit the general increase in permeability of infected cells to a range of substrates(18, 59, 61, 62) , they cannot necessarily be considered to act by causing lactic acid accumulation.

  
Table: Inhibition of L-lactate uptake into control and parasitized erythrocytes by other monocarboxylates

The competing substrate and 0.5 mM [C]lactate were added simultaneously and transport terminated by centrifugation after 15 s at 0 °C for parasitized cells and 90 s at 25 °C for control cells. Data are presented as the means ± S.E. of four separate experiments for which mean parasitemias (percent trophozoites) varied from 66 to 69%.


  
Table: Inhibition of L-lactate uptake into control and parasitized erythrocytes by various inhibitors

The uptake of 0.5 mML- or D-[C]lactate was measured as described in Table I. Inhibitors were added at the concentrations shown 1 min before the addition of lactate. The pH gradient was shown to be unaffected by any of the inhibitors used. All data are expressed as mean values ± S.E. for n experiments for which mean parasitemias (percent trophozoites) varied from 62 to 76%. The statistical significance of differences between inhibition of control and parasitized cells was calculated using a two-tailed Student's t test (*p < 0.01,**p < 0.001).


  
Table: Comparison of the inhibition by various compounds of L-lactate and chloride transport into parasitized erythrocytes

The uptake of either 0.6 mML-[C]lactate (30 s) or Cl (10 s) into parasitized cells at 0 °C was measured in the presence and absence of inhibitor at the concentration shown. Both lactate and Cl uptakes were performed on the same cell preparations (parasitemias ranged from 48 to 72%). Control rates of transport assuming linearity of uptake with time and a deadtime of 8 s were 0.84 ± 0.06 and 1.31 ± 0.03 nmol/µl/min for lactate and chloride, respectively. The ratio of the inhibition of lactate to Cl transport was used to assess significant differences in inhibitor potency using a paired Student's t test (*p < 0.05,**p < 0.02,***p < 0.01).



FOOTNOTES

*
This work was supported by a Medical Research Council Collaborative Research Studentship with The Wellcome Foundation Ltd. and by a project grant from The Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Microbiology, Monash University, Clayton, Victoria 3168, Australia.

To whom correspondence should be addressed: Dept. of Biochemistry, School of Medical Sciences, University of Bristol, Bristol Walk, Bristol BS8 1TD, U. K. Tel.: 44-272-288592; Fax: 44-272-288274.

The abbreviations used are: CHC, -cyano-4-hydroxycinnamate; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonate; DBDS, 4,4`-dibenzamidostilbene-2,2`-disulfonate; DNDS, 4,4`-dinitrostilbene-2,2`-disulfonate; PCMBS, p-chloromercuribenzenesulfonate; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; DMO, dimethyloxazolidine-2,4-dione.


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