Functional state of the plasma membrane Ca²⁺ pump in Plasmodium falciparum-infected human red blood cells

Abstract

1. The active Ca²⁺ transport properties of malaria-infected, intact red blood cells are unknown. We report here the first direct measurements of Ca²⁺ pump activity in human red cells infected with Plasmodium falciparum, at the mature, late trophozoite stage.

2. Ca²⁺ pump activity was measured by the Co²⁺-exposure method adapted for use in low-K⁺ media, optimal for parasitised cells. This required a preliminary study in normal, uninfected red cells of the effects of cell volume, membrane potential and external Na⁺/K⁺ concentrations on Ca²⁺ pump performance.

3. Pump-mediated Ca²⁺ extrusion in normal red cells was only slightly lower in low-K⁺ media relative to high-K⁺ media despite the large differences in membrane potential predicted by the Lew-Bookchin red cell model. The effect was prevented by clotrimazole, an inhibitor of the Ca²⁺-sensitive K⁺ (K_Ca) channel, suggesting that it was due to minor cell dehydration.

4. The Ca²⁺-saturated Ca²⁺ extrusion rate through the Ca²⁺ pump (V_max) of parasitised red cells was marginally inhibited (2-27 %) relative to that of both uninfected red cells from the malaria-infected culture (cohorts), and uninfected red cells from the same donor kept under identical conditions (co-culture). Thus, Ca²⁺ pump function is largely conserved in parasitised cells up to the mature, late trophozoite stage.

5. A high proportion of the ionophore-induced Ca²⁺ load in parasitised red cells is taken up by cytoplasmic Ca²⁺ buffers within the parasite. Following pump-mediated Ca²⁺ removal from the host, there remained a large residual Ca²⁺ pool within the parasite which slowly leaked to the host cell, from which it was pumped out.

Calcium ions are required at two critical stages during the intraerythrocytic asexual life-cycle of P. falciparum: for a few seconds during the invasion of the red cell by the parasite, and continuously for normal parasite development (Wasserman et al. 1982; Tanabe, 1990). The total calcium content of parasitised cells increases with parasite maturation far above the negligible levels observed in uninfected normal red cells (Bookchin et al. 1980). Evidence suggests that this increase is confined to internal parasite compartments (Lee et al. 1988; Tanabe, 1990; Kramer & Ginsburg, 1991; Desai et al. 1996). Calcium homeostasis of malaria-infected red cells has been the subject of intense research in recent years, with substantial advances in our knowledge of stage-related changes in the passive Ca²⁺ permeability of the membrane (Kramer & Ginsburg, 1991; Desai et al. 1996; Staines et al. 1999). Nothing is known, however, about the performance of the plasma membrane Ca²⁺ pump (PMCA) in intact, parasitised red cells. The only reported measurements of Ca²⁺-Mg²⁺-ATPase activity were done in isolated membranes of murine red cells infected with P. chabaudi (Tanabe, 1990). The results suggested that the pump enzyme was inhibited by about 30 % relative to uninfected controls. There have been no further studies in other Plasmodii infections or on the functional state of the pump in the intact parasitised cell. It is important to bear in mind that the PMCA is a multi-regulated transporter (Schatzmann, 1982) and that ATPase activity in isolated membranes may not reflect the performance of the pump in the intact cell.

We report here the first direct measurements of PMCA-mediated Ca²⁺ extrusion in intact human red cells infected with P. falciparum at the late trophozoite stage. Assessment of Ca²⁺ pump function in intact, uninfected red cells is routinely done by the Co²⁺-exposure method (Dagher & Lew, 1988). However, this method had to be modified to obtain reliable Ca²⁺ flux measurements in parasitised cells. This required a preliminary feasibility study in normal uninfected red cells.

METHODS

Solutions

Solution A (low-K⁺, plasma-like medium) contained (mM): KCl, 3; NaCl, 140; MgCl₂, 0.2; Hepes-Na (pH 7.4-7.5 at 37°C), 10; and Na-EGTA, 0.1. Solution B was the same as solution A but without Na-EGTA. Solution C (‘clamp’ medium) contained (mM): KCl, 80; NaCl, 70; MgCl₂, 0.15; and Hepes-Na (pH 7.4-7.5 at 37°C), 10. Stock solutions of ⁴⁵CaCl₂ were prepared at concentrations of 40–100 mM and with ⁴⁵Ca²⁺ specific activity between 10⁷ and 10⁸ c.p.m. (μmol)⁻¹. A23187 was dissolved in ethanol, as a 2 mM stock solution. CoCl₂ was prepared as a stock solution (100 mM) in distilled water. Sodium orthovanadate was prepared as a stock solution (100 mM) in solution B. Clotrimazole was prepared as a stock solution (10 mM) in DMSO.

Parasite cultures

The P. falciparum cloned strain A4 (kind gift of B. C. Elford, Institute of Molecular Medicine, Oxford, UK), derived from the P. falciparum ITO4 line (Berendt et al. 1989), was cultured in human red blood cells (type O) by standard methods (Trager & Jensen, 1976), under a low-oxygen atmosphere (1 % O₂, 3 % CO₂, 96 % N₂). The culture medium was RPMI-1640, supplemented with D-glucose (10 mM), glutamine (2 mM), Hepes (40 mM), gentamicin sulphate (25 mg l⁻¹) and 8.5 % (v/v) pooled human serum. Parasites were synchronised by a combination of sorbitol haemolysis (Lambros & Vanderberg, 1979) and gelatin flotation (Jensen, 1978). All experiments with malaria-infected erythrocytes were carried out using red blood cells infected with P. falciparum at the late trophozoite stage (36-40 h post-invasion). Parasitaemia was assessed by microscopic inspection of Giemsa-stain thin blood smears. Cell counts were estimated on suspension samples (5 μl) fixed in formalin (100 μl) using an improved Neubauer counting chamber.

Use of the Co²⁺-exposure method to measure PMCA-mediated Ca²⁺ transport in P. falciparum-infected red cells

In the Co²⁺-exposure method (Dagher & Lew, 1988; Lew & García-Sancho, 1989) a uniform Ca²⁺ load is rapidly induced in a red cell population by means of the ionophore A23187. After 1–2 min, Co²⁺ is added to the cell suspension to instantly block ionophore-mediated Ca²⁺ transport, thereby exposing uphill Ca²⁺ extrusion by a Ca²⁺-saturated pump (V_max). During this procedure the cells become permeabilised to Ca²⁺ and Mg²⁺ via the ionophore, and indirectly to K⁺, by activation of their K_Ca channels (Gardos, 1958; Lew & Ferreira, 1978). In low-K⁺, plasma-like media, the cells would hyperpolarise, lose KCl and water, and acidify (Lew & Bookchin, 1986; Freeman et al. 1987). These changes in cell volume, pH, membrane potential and ion content, resulting from the increased ion permeabilities, are prevented by a ‘homeostatic clamp’ obtained by setting the external Mg²⁺ and K⁺ concentrations at electrochemical equilibrium with the intracellular ions, to minimise net ion fluxes other than those of Ca²⁺ (Dagher & Lew, 1988; Lew & García-Sancho, 1989). Application of this procedure to P. falciparum-infected red cells is not feasible. Parasitised cells undergo marked changes in their membrane transport properties due to the early appearance of new permeation pathways (NPPs) (Ginsburg et al. 1986; Lee et al. 1988; Kirk et al. 1991, 1992). Although NPPs are anion selective, they also increase the Na⁺ and K⁺ permeabilities resulting in the rapid dissipation of monovalent cation gradients. This predisposes the red cells infected with late trophozoites to rapid haemolysis in high-K⁺ media, such as those used for clamp conditions in the standard Co²⁺-exposure method. A suspension of infected cells, however well synchronised, represents an heterogenous mix of cells in different homeostatic states, which is impossible to clamp in a single suspending medium. Since parasitised cells retain optimal volume stability in low-K⁺ media, such as RPMI media used for in vitro cultures, it would be desirable to measure pump activity in similar low-K⁺ media. The question would then be whether Ca²⁺ pump performance would be comparable in and out of clamp conditions, and whether the predicted changes in membrane potential, cell volume, ion content and pH would affect the measurements in ways that precluded proper assessment of pump function. To answer these questions it was necessary to compare Ca²⁺ pump function in standard clamp conditions and in low-K⁺ media. This comparison was performed in fresh, uninfected red cells. Besides the value of the results for the general characterisation of Ca²⁺ pump properties, the study provided the information required for the Co²⁺-exposure method to be applied to P. falciparum-infected cells. It is worth pointing out that the only alternative to the Co²⁺-exposure method, i.e. measurement of pump-mediated Ca²⁺ extrusion after ionophore wash-out (Pereira et al. 1993), cannot be applied to P. falciparum-infected cells because these cells could not withstand the required low-temperature washes.

Experiments with fresh, uninfected red cells

Venous blood from healthy volunteers was drawn into heparinised syringes after written consent. The cells were washed 3 times by centrifugation (2500 g, 5 min) and resuspension in 6–8 volumes of solution A to remove Ca²⁺ loosely bound to the outer cell surface (Harrison & Long, 1968). The cell pellet was divided into two aliquots. One aliquot was washed 3 more times in solution B to remove EGTA from the medium, and the other aliquot 3 more times in solution C. After each spin, the supernatant and the top cell layer containing white cells and platelets were removed. After the washes the cells were suspended at ∼10 % haematocrit in solution B or C, supplemented with inosine (10 mM) and the additives indicated in each case. The haematocrit of cell suspensions was estimated from spectrophotometric haemoglobin (Hb) measurements at 540 nm by the cyanmethaemoglobin method. In the experiments where clotrimazole was used as a specific inhibitor of the red cell K_Ca channels (Alvarez et al. 1992), clotrimazole was added to a final concentration of 10 μM in the cell suspension, sufficient to inhibit over 99 % of K_Ca-mediated K⁺ fluxes (Brugnara et al. 1994).

Experiments with P. falciparum-infected red cells, cohort and co-cultured cells

Cells from cultures were separated by centrifugation (400 g, 10 min) on Percoll diluted with ×10 phosphate-buffered saline and water to a density of 1.090 (∼66 % v/v) and osmolality of 320 mosmol (kg H₂O)⁻¹ (Kirk et al. 1996). Suspensions with 50–96 % parasitaemia were obtained by this method. Uninfected red blood cells from the same culture (cohorts) were harvested from the cell pellet under Percoll. The cohort pellet had < 5 % parasitised red cells, mainly young, immature parasites. Uninfected red cells from the same donors were incubated in parallel (co-cultured cells) under identical conditions, for at least 48 h prior to the experiment. These cells and cohorts were used as controls.

Since the Co²⁺-exposure protocol had to be carried out consecutively for each of the different conditions, it was important to ensure that the various suspensions of Percoll-harvested parasitised cells reached the flux assay in optimal state. Percoll-harvested parasitised cells were washed twice by centrifugation (600 g, 5 min) and resuspension in culture medium without serum to remove residual Percoll; after the washes the cells were resuspended in culture medium (< 0.5 % haematocrit) and kept at room temperature, under low P_O2, until use. This procedure gave comparable results for the first and last processed control groups within each experiment, and preserved the normal morphology of the parasitised cells without extra lysis. Haematocrits were estimated from cell counts per unit volume (see below). All suspensions with Percoll-harvested parasitised cells were inspected to assess the morphological condition of the infected cells. Five to 10 μl of fresh, unfixed cell suspensions in RPMI were placed between slide and coverslip, observed by phase contrast under oil at ×1000, and video-recorded. If there was no evidence of Brownian motion of haemozoin crystals within the parasites’ food vacuoles the sample was discarded.

Measurement of PMCA-mediated Ca²⁺ extrusion in parasitised red cells, cohorts, co-cultured cells, and fresh, uninfected red cells

Immediately before use the cells were washed twice in solution B and resuspended (3-10 % haematocrit) in solution B with 10 mM inosine and 5 mM glucose. ⁴⁵CaCl₂ was added to 1.5-2.0 ml aliquots of cell suspensions to a final Ca²⁺ concentration in the suspensions ([Ca_T]_s) of 124–170 μM (specific details in the corresponding figure legends). The low-haematocrit conditions chosen in these experiments ensured minimal dilution of the original ⁴⁵Ca²⁺ specific activity by the parasites’ endogenous cold Ca²⁺ pools. At least 10⁸ parasitised cells were used for each condition. The suspensions were pre-incubated at 37°C for 5–10 min under magnetic stirring before the Co²⁺-exposure protocol was carried out. At time (t)= 0, the ionophore A23187 was added from a concentrated stock solution to final concentrations intended to ensure rapid ⁴⁵Ca²⁺ redistribution to equilibrium in both infected and uninfected red cells. Normal, uninfected human red cells are virtually Ca²⁺ free (Harrison & Long, 1968; Bookchin & Lew, 1980; Engelmann & Duhm, 1987) but P. falciparum parasites contain a substantial endogenous Ca²⁺ pool which increases the mean total intracellular calcium concentration ([Ca_T]_i) of the infected red cells by more than tenfold. Bookchin et al. (1980) reported a [Ca_T]_i of 52 μmol (l cells)⁻¹ in a 60 % parasitised cell suspension. Ionophore-induced Ca²⁺ permeabilisation mixes all membrane-bound compartments containing Ca²⁺ pools (Bookchin et al. 1980). At t = 1-2 min, CoCl₂ was added to the suspension to a concentration of 400 μM and uphill Ca²⁺ extrusion was followed for 6–8 min, thus minimising both the ATP-depleting effect caused by the pump ATPase activity (Dagher & Lew, 1988; García-Sancho & Lew, 1988) and the toxic effects of long-term parasite exposure to ionophore (Krungkrai & Yuthavong, 1983). Samples (50 μl) were taken throughout the procedure at the times indicated in the figures for measurements of [Ca_T]_i, and processed as reported before (Dagher & Lew, 1988; Tiffert & Lew, 1997). In the experiments with orthovanadate, sampling for [Ca_T]_i was continued for an extended period of 20 min. Ionophore and pump-mediated Ca²⁺ fluxes were estimated from the slope of the curves reporting [Ca_T]_i as a function of time. Initial Ca²⁺ extrusion after Co²⁺ addition is usually linear. It represents the maximal Ca²⁺ extrusion rate through a Ca²⁺-saturated pump; the initial slope reports the pump V_max and was estimated by linear regression.

For consistency with data in the literature and for internal comparability, Ca²⁺ fluxes in fresh, uninfected cells, from suspensions in which haematocrits were estimated from spectrophotometric Hb measurements, were expressed in units of millimoles per 340 grams of haemoglobin per hour (mmol (340 g Hb)⁻¹ h⁻¹). In the experiments with cultured cells, from suspensions in which haematocrits were estimated from cell counts, Ca²⁺ fluxes were expressed in units of millimoles per 10¹³ cells per hour (mmol (10¹³ cells)⁻¹ h⁻¹). Within a 10–15 % variation, 340 g Hb may be assumed to represent the 10¹³ cells present in 1 l of normal volume, packed red blood cells. When estimated from cell counts per unit volume, haematocrits were calculated as fractions of 10¹³ cells per litre, assumed to represent a haematocrit of 100 %.

Use of the red cell model to estimate the value of red cell homeostatic variables

Estimates of the approximate value of relevant homeostatic variables (membrane potential, cell volume and pH), and analysis of the changes induced by the different conditions set up in the experiments with fresh uninfected cells, were done using the Lew-Bookchin red cell model (Lew & Bookchin, 1986). For the range of conditions studied here, this model was shown to predict the value of those variables with a precision level of 5–10 % (Freeman et al. 1987; Tiffert et al. 1993; Raftos & Lew, 1995; Etzion et al. 1996). The simulations followed the experimental protocols applied here and the relevant values obtained in each case are reported in Results.

The Lew-Bookchin red cell model is available as a free-standing program for PC platforms from http://www.physiol.cam.ac.uk/staff/lew/index.htm

Materials

All chemicals were analytical reagent quality. Plasmagel (Bellon, Neuilly Sur Seine, France) was a kind gift from Barry C. Elford, Institute of Molecular Medicine, Oxford (UK). EGTA, Hepes, glucose, inosine, clotrimazole, sodium orthovanadate, CoCl₂, DMSO, RPMI-1640 medium (R0883), gentamicin sulphate, glutamine and formalin solution were from Sigma-Aldrich Co. (UK). A23187 was from Calbiochem-Novabiochem (UK) Ltd. CaCl₂, MgCl₂, NaCl and KCl were from FSA Laboratory Supplies (Loughborough, UK). ⁴⁵Ca²⁺ was from Amersham International plc (Little Chalfont, UK). PBS was from Life Technologies Ltd (UK). Percoll was from Amersham Pharmacia Biotech. (Sweden). Human blood and serum used in cultures were from the Eastern Anglia Blood Centre, Cambridge (UK).

RESULTS

Comparison of PMCA performance in uninfected red cells in and out of homeostatic clamp conditions

In the experiment illustrated in Fig. 1, typical of four others with similar results, fresh normal red cells were suspended either in low-K⁺ solution B, or in high-K⁺, clamp-solution C, with or without clotrimazole. Addition of the ionophore A23187 caused rapid loading of the cells with [⁴⁵Ca_T]_i to a level of ∼600 μmol (340 g Hb)⁻¹, which was similar for both media. This Ca²⁺ load fully activates the K_Ca channels of the cells, effectively increasing their K⁺ permeability by over three orders of magnitude (Lew & Ferreira, 1978). In the experiment of Fig. 1, the Ca²⁺ pump V_max values were 15.8 and 16.3 mmol (340 g Hb)⁻¹ h⁻¹ for control clamp conditions, without and with clotrimazole, respectively, indicating that clotrimazole per se has no effect on pump-mediated fluxes. In low-K⁺ media, the Ca²⁺ pump V_max values were 14.9 and 16.4 mmol (340 g Hb)⁻¹ h⁻¹ in the absence and presence of clotrimazole, respectively. In the four experiments of this series, the V_max values of the Ca²⁺ pump in low K⁺ were 3.5-7.0 % lower than those in clamp conditions, and 6.5-9.0 % lower than those in low K⁺ with clotrimazole. Though hardly significant within each experiment, the pump inhibitory effects of low-K⁺ media were present in all experiments. The effect was prevented by clotrimazole, a K_Ca channel inhibitor, suggesting that it was due to minor cell dehydration caused by the activation of the K_Ca channels in the low-K⁺ medium.

Figure 1. Measurement of Ca²⁺ pump-mediated Ca²⁺ extrusion from intact fresh red cells in both high- and low-K⁺ media: effects of clotrimazole.

Red cells from freshly drawn blood were suspended at 10 % haematocrit in either high-K⁺, clamp-solution C (circles), or in low-K⁺ solution B (triangles and squares), both supplemented with 10 mM inosine, and with (open circles, squares) or without (filled circles, triangles) clotrimazole (10 μM). All cell suspensions contained ⁴⁵Ca²⁺. The ⁴⁵Ca²⁺ concentration added to the cell suspensions ([Ca_T]_s) was ≈150 μM. Arrows indicate additions of ionophore A23187 (t = 0, 10 μM) and CoCl₂ (t = 2 min, 400 μM). The V_max values correspond to the slope of the linear regression lines through the indicated points, chosen by eye for each curve.

For the low-K⁺ conditions of these experiments, with the Cl⁻ permeability rate-limiting to net KCl loss and dehydration, the red cell model predicted membrane potential hyperpolarisation to about -90 mV, less than 10 % dehydration within the 8 min duration of the experiments and insignificant cytoplasmic acidification. The results in Fig. 1 show that Ca²⁺ pump performance was not significantly affected by any of the following: large changes in membrane potential and external Na⁺/K⁺ concentrations, isotonic cell dehydration (< 10 %), or by the associated minor changes in cell and medium pH (Freeman et al. 1987). Moreover, the different conditions had no effect on ionophore-induced Ca²⁺ influx or on the ionophore-induced equilibrium distribution of Ca²⁺ between cells and medium (Fig. 1). Thus, the Co²⁺-exposure protocol used here may be safely applied to investigate pump-mediated Ca²⁺ extrusion from uninfected and infected cells in non-clamp, low-K⁺ media, as long as the K_Ca channels are blocked or the measurements completed before significant cell dehydration.

PMCA performance in P. falciparum-infected red cells

PMCA function in infected cells was explored in low-K⁺, non-clamp media only. Cell counts before and after the Co²⁺-exposure protocol showed less than 5 % haemolysis for both infected and uninfected cells under these conditions, comparable to that observed under clamp conditions. In the experiment of Fig. 2, the ionophore concentration was the same for parasitised and cohort cell suspensions (10 μM). The pattern of changes in [Ca_T]_i with time was markedly different. The main difference was in the ionophore-induced Ca²⁺ influx which was relatively slow in the parasitised cells. It was clear that an ionophore concentration sufficient to induce Ca²⁺ equilibration by 1 min in uninfected cells failed to induce a significant Ca²⁺ load in infected cells. This reduced ionophoric effect has been observed before (Krungkrai & Yuthavong, 1983). Its possible origin is considered in Discussion.

Figure 2. Comparative response of parasitised and cohort cells to the Co²⁺-exposure protocol using identical ionophore concentrations.

Cells were suspended in low-K⁺ solution B with 10 mM inosine and 5 mM glucose. Ionophore A23187 and Co²⁺ were added as described in Fig. 1, except for the timing of Co²⁺ addition which was 1 min after addition of the ionophore. Parasitaemia was 87 % in the parasitised cell suspension and ≈4.5 % (mostly immature, ring-stage parasites) in the cohort cell suspension. The ⁴⁵Ca²⁺ concentration added to the cell suspensions ([Ca_T]_s) was 124 μM. The haematocrits of parasitised and cohort cell suspensions were 3.9 and 6.2 %, respectively, relative to 100 %= 10¹³ cells l⁻¹. V_max values were calculated from the slope of the linear regression lines through the indicated points, chosen by eye for each curve. Note that the parasitised cells show a slow ionophore-induced Ca²⁺ influx, a limited Ca²⁺ load, a large apparent V_max inhibition and a slightly higher residual calcium content than the cohorts.

Further experiments indicated that doubling of the ionophore concentration was required to elicit comparable loading patterns between infected and uninfected cells without causing additional haemolysis. Figure 3 shows the results obtained using a higher ionophore concentration (20 μM) in the parasitised cell suspensions. Parasitaemia in the parasitised cell suspensions was 50 % in Fig. 3a and 96 % in Fig. 3b. The results are representative of two other similar experiments, with 56 and 91 % infected cells, respectively. Ionophore-induced Ca²⁺ loads reached steady [Ca_T]_i levels before Co²⁺ addition in all conditions. Estimates of the minimal net Ca²⁺ influx induced by the ionophore may be obtained from the Ca²⁺ gained by the cells in the first 20 s following ionophore addition. These varied between 100 and 200 mmol (10¹³ cells)⁻¹ h⁻¹, far higher than the maximal pump V_max in any condition (see below). Thus, the near-plateau [Ca_T]_i levels attained must have been close to the Ca²⁺ equilibrium distributions induced by the ionophore. The effect of vanadate is illustrated in Fig. 3b. As with normal human red cells (Tiffert & Lew, 1997), 1 mM vanadate fully inhibited uphill Ca²⁺ extrusion from parasitised cells. The similarity between the Ca²⁺ load induced by the ionophore and the level of [Ca_T]_i sustained in the presence of vanadate confirms that the 2 min [Ca_T]_i load is close to the ionophore-induced equilibrium. In each experiment, the Ca²⁺ load at equilibrium was higher in the parasitised cells than in the uninfected controls. The higher the parasitaemia the higher the [Ca_T]_i load at equilibrium (compare Fig. 3a with B). Fig. 3b also shows that both parasitised and uninfected cells responded with increased loads to increased [Ca_T]_s.

Figure 3. Comparative response of parasitised, co-cultured and cohort cells to the Co²⁺-exposure protocol, using a higher ionophore concentration in parasitised cells: Ca²⁺ pump V_max and effect of vanadate.

Cells were suspended in low-K⁺ solution B with 10 mM inosine and 5 mM glucose. Ionophore A23187 and Co²⁺ were added as described in Fig. 1. The ionophore concentration was 20 μM in the parasitised cell suspensions and 10 μM in co-cultured and cohort cell suspensions. A, parasitaemia in the parasitised cell suspension was 50 %; [Ca_T]_s= 150 μM in parasitised and co-cultured cell suspensions; haematocrits of parasitised and co-cultured cells were 3.72 and 4.54 %, respectively. Pump V_max values (slope ±s.e.m.) were 11.4 ± 0.5 mmol (10¹³ cells)⁻¹ h⁻¹ for parasitised cells, and 15.5 ± 0.4 mmol (10¹³ cells)⁻¹ h⁻¹ for co-cultured cells. The residual Ca²⁺ extrusion rate from parasitised cells, estimated by linear regression through the points between 12–20 min, was about 0.3 mmol (10¹³ cells)⁻¹ h⁻¹, equivalent to 0.6 mmol (10¹³ cells)⁻¹ h⁻¹, when corrected to 100 % parasitaemia. B, parasitaemia in the parasitised cell suspension was 96 %; parasitaemia in the cohort cell suspension was < 5 % (immature, ring-stage parasites). [Ca_T]_s for each condition is indicated above the corresponding ionophore-induced equilibrium [Ca_T]_i level. The Ca²⁺-loading stage for the condition with vanadate was omitted from the figure; it showed a similar equilibrium load as the condition with [Ca_T]_s= 169 μM. The vanadate concentration in the medium was 1 mM. Haematocrits for each of the six curves were (from top to bottom): 4.3 (vanadate), 4.8, 4.4, 7.1, 6.5 and 7.7%. The V_max values obtained for the five curves showing uphill Ca²⁺ extrusion were (from top to bottom, slope ±s.e.m.): 11.70 ± 0.73; 10.91 ± 0.54; 9.08 ± 0.64; 14.38 ± 0.32; and 12.50 ± 0.26 mmol (10¹³ cells)⁻¹ h⁻¹. Note the lower coefficient of variation of the last two curves corresponding to uninfected cells. The residual Ca²⁺ extrusion rate from parasitised cells is reported here for the condition with [Ca_T]_s= 150 μM; the linear regression through points between 8–15 min gave a rate of about 1.2 mmol (10¹³ cells)⁻¹ h⁻¹.

The V_max of the Ca²⁺ pump was estimated from the linear Ca²⁺ extrusion rate immediately after addition of Co²⁺. The pump V_max of infected and uninfected cells varied between 8.1 and 15.5 mmol (10¹³ cells)⁻¹ h⁻¹ in the four experiments of this series. These values fell well within the range of normal variation observed before in fresh human red cells (Dagher & Lew, 1988; Tiffert et al. 1993). The V_max value in cohort cells was lower than that in co-cultured controls (Fig. 3b). The V_max values in the infected cell samples were 2–27 % lower than those in cohort and co-cultured samples in the different experiments.

Another systematic and more subtle difference in V_max between infected and uninfected cells concerned the scatter of the experimental points. The coefficients of variation of the linear regression slopes obtained from uninfected cells were usually less than 2.5 %, whereas those from infected cells varied between 4.5 and 7.5 % (5.0-7.1 % in the experiments of Fig. 3b). Also, the choice of the number of points to use for V_max estimates by linear regression was less clear in infected cells. This was due to the marked increase in Ca²⁺ retention within the parasitised cells relative to uninfected controls (Fig. 3). In the experiment with 96 % parasitaemia (Fig. 3b), the level of [Ca_T]_i at which Ca²⁺ efflux slowed down after the initial rapid Ca²⁺ extrusion period was between 700 and 900 μmol (10¹³ cells)⁻¹ in infected cells, and about 200 μmol (10¹³ cells)⁻¹ in uninfected controls. In the two experiments with lower parasitaemia, the difference was smaller but still substantial. The residual Ca²⁺ extrusion rate from the 96 % parasitaemia suspensions was about 1.2 mmol (10¹³ cells)⁻¹ h⁻¹ (Fig. 3b) whereas that from the suspension with 50 % parasitaemia was 0.6 mmol (10¹³ cells)⁻¹ h⁻¹ (corrected to 100 % parasitaemia, Fig. 3a).

DISCUSSION

The present results report the first direct measurements of Ca²⁺ pump performance in P. falciparum-infected, intact red cells. The preliminary investigation (Fig. 1) showed that the activity of the Ca²⁺ pump in uninfected, normal red cells was minimally affected by large variations in membrane potential and external Na⁺ and K⁺ concentrations, or by minor cell dehydration. This comparability of pump V_max rates established the feasibility of measuring Ca²⁺ pump V_max by the Co²⁺-exposure method outside the usual clamp constraining conditions, provided the measurements were performed in the presence of K_Ca channel blockers, or completed before significant cell dehydration, as in the present study. Comparison of results between normal, infected, cohort and co-cultured cells revealed substantial differences in the Ca²⁺-permeabilising efficiency of the ionophore, the [Ca_T]_i load at equilibrium and the Ca²⁺-desaturation pattern after Co²⁺ addition.

To interpret these results it is necessary to analyse the events which take place in parasitised cells during the Co²⁺-exposure protocol. When ionophore is added to isolated cells in suspension, it partitions extremely rapidly and reversibly into all plasma and organelle membranes, with partition ratios membrane/medium of > 60 (Lew & Simonsen, 1981). In the membranes, the ionophore performs electroneutral M²⁺:2H⁺ exchange, driving the distribution of divalent cations (M²⁺; mainly Ca²⁺ and Mg²⁺) across each membrane towards an equilibrium given by [M²⁺]_i/[M²⁺]_o= ([H⁺]_i/[H⁺]_o)² (Pressman, 1976). The magnitude of the ionophoric effect is an exponential function of the ionophore concentration in the membrane (Lew & Simonsen, 1980; Simonsen & Lew, 1980). Thus, the more membrane area available for ionophore distribution the more diluted its ionophoric effect would be at each total ionophore concentration. This provides a clear and simple explanation for the results observed in Fig. 2, where an ionophore concentration sufficient to induce rapid Ca²⁺ equilibration across the membrane of uninfected cells, failed to do so in a suspension of infected cells with comparable haematocrit and 87 % parasitaemia (Fig. 2). The tentative explanation from the analysis above is that the extra membrane area provided by parasites effectively diluted the ionophore concentration in parasite and host cell membranes relative to its concentration in the plasma membrane of the uninfected cells, causing an exponential reduction in the ionophoric effect. Thus, increasing the ionophore concentration in parasitised cells, to levels which would normally destabilise uninfected red cell membranes and enhance their lysis, simply increased the ionophoric effect in the parasitised cell without further lysis (Fig. 3). Krungkrai & Yuthavong (1983) reported that the capacity of the ionophore A23187 to induce Ca²⁺ uptake was much reduced in mice red cells infected with P. berghei relative to uninfected red cells, despite a substantial increase in ionophore uptake by the infected cells. The reduced ionophoric capacity was attributed to abnormal interactions between the ionophore and the membrane of the infected cells. However, their observation of increased ionophore uptake in infected cells is precisely what would be expected from the increased membrane area. The consequent dilution effect provides a simple explanation of all the experimental results in terms of known ionophore properties.

At adequate ionophore concentrations, both parasitised and uninfected red cells attained Ca²⁺ equilibration in less than 2 min (Fig. 3). The total Ca²⁺ content at equilibrium was substantially higher in parasitised cells than in uninfected cells. To interpret the significance of this difference it is necessary to analyse the distribution of Ca²⁺ induced by the ionophore in uninfected controls and in infected cells. The equilibrium distribution of free ionised Ca²⁺ between cells and medium in uninfected cells is given by:

(1)

where r represents the proton ratio set by the operation of the Jacobs-Stewart mechanism (Jacobs & Stewart, 1947), usually about 1.4 (r²∼2) in clamp conditions (Lew & García-Sancho, 1989). The Lew-Bookchin model predicts that outside clamp conditions the cells would dehydrate by less than 6 % by the time the ionophore-induced equilibrium is attained (< 2 min; Fig. 1), and that r would increase also by less than 6 %. Thus, the clamp value of r may be considered largely conserved in non-clamp conditions as long as cell dehydration does not exceed 6 %.

The Ca²⁺ concentration added to the cell suspension ([Ca_T]_s) becomes distributed between cells ([Ca_T]_i) and medium ([Ca²⁺]_o) according to:

(2)

where Hct is expressed here as the cell volume fraction (haematocrit/100). At high-Ca²⁺ loads, cytoplasmic Ca²⁺ buffering in human red cells is well approximated by the equation (Ferreira & Lew, 1977; Tiffert & Lew, 1997):

(3)

where α (fraction of total cell calcium which is in ionised form in cell water) is approximately 0.3 (Tiffert & Lew, 1997). It is now possible to estimate the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and [Ca²⁺]_o, at equilibrium, in the uninfected cells using: (i) eqns (2) and (3); (ii) measured [Ca_T]_i; (iii) known [Ca_T]_s and Hct; and (iv) the approximate value of α. For instance, the values of [Ca_T]_s, [Ca_T]_i and Hct for the co-cultured cells in Fig. 3b were: 160 μM, 780 μmol (10¹³ cells)⁻¹ and 0.065, respectively. With these values, [Ca²⁺]_o, from eqn (2), renders 116 μM; and [Ca²⁺]_i, from eqn (3), gives 234 μM. The ratio [Ca²⁺]_i/[Ca²⁺]_o is thus approximately 2.0. From eqn (1), we calculate r²= 2, which corresponds to the clamp value of r². This coincidence, repeated in all the experiments with uninfected cells, confirms that the normal proton concentration ratio persists largely unchanged in non-clamp conditions, at least for 2 min after ionophore addition, as expected from the predicted negligible extent of dehydration (< 6 %).

To analyse the equilibrium distribution of Ca²⁺ in the infected cells it is necessary to consider that the total measured cell Ca²⁺ content ([Ca_T]_i) is now divided between host ([Ca_T]_H) and parasite ([Ca_T]_P). Thus:

(4)

where f_V is the mean volume fraction of red cell cytosol remaining in the parasitised cell sample under study. As defined, this comprises the mean residual volume fraction of host cell cytosol in the infected cells as well as the full volume fraction of uninfected cells in the population. Assuming that cytoplasmic Ca²⁺ buffering in the host cells is not altered, eqn (3) becomes:

(5)

where [Ca²⁺]_H represents the free ionised Ca²⁺ concentration in the host cytosol. Equation (1) becomes:

(6)

For precise solutions of the four unknowns [Ca²⁺]_H, [Ca²⁺]_o, [Ca_T]_H and [Ca_T]_P, using eqns (2), (4), (5) and (6), it would have been necessary to measure r and f_V. This was outside the scope of the present investigation. For the analysis below it is sufficient to rely on approximate estimates of r and test alternative values of f_V. Recent non-invasive fluorimetric pH measurements by Wünsch et al. (1998) have indicated that host cell pH is about 7.12 ± 0.02, very near normal. In the absence of more detailed stage-related data it will be assumed that the normal value of r, of about 1.4, is conserved in the parasitised cells throughout the parasite's asexual cycle.

In the suspension of parasitised cells with [Ca_T]_s= 150 μM (Fig. 3b), for instance, the mean [Ca_T]_i at equilibrium was 1150 μmol (10¹³ cells)⁻¹, with a Hct of 0.071. From eqns (2), (6) and (5), we successively calculate [Ca²⁺]_o= 73.6 μM, [Ca²⁺]_H= 147 μM (using r²= 2) and [Ca_T]_H= 490 μmol (10¹³ cells)⁻¹ (using α= 0.3). Assuming f_V= 0.4, eqn (4) renders [Ca_T]_P= 1590 μmol (10¹³ cells)⁻¹. If f_V= 0.6, then [Ca_T]_P= 2140 μmol (10¹³ cells)⁻¹. These rough estimates indicate that in suspensions of red cells infected with P. falciparum at the mature trophozoite stage, in conditions of ionophore-induced Ca²⁺ equilibration, most of the ionophore-induced [Ca_T]_i load is within the parasite ((1 –f_V)[Ca_T]_P >> f_V[Ca_T]_H), the total Ca²⁺ concentration of the parasite is much higher than that of the host ([Ca_T]_P >> [Ca_T]_H), and the real differences in total Ca²⁺ concentrations between host and parasite are much larger than apparent from the observed differences between infected and uninfected cells (Fig. 3b). Since the ionophore permeabilises to Ca²⁺ all membrane-bound compartments of the host-parasite system, the elevated total Ca²⁺ concentration within the parasite indicates either that the parasite has a large Ca²⁺-binding capacity associated with cytoplasmic Ca²⁺-buffering systems, or that large pH gradients persist across internal parasite membranes, causing an elevated Ca²⁺ partition within acidic compartments (according to eqn (1)), or both. The pH of the parasite cytoplasm is similar to that of the host cell (Bosia et al. 1993; Wünsch et al. 1998). Thus, the concentration of free Ca²⁺ would not be expected to differ much between host and parasite cytosols. This analysis, together with the results obtained so far, establish the feasibility of investigating the cytoplasmic Ca²⁺ buffering of P. falciparum parasites in situ, within their host red cells.

Pump-mediated Ca²⁺ extrusion from parasitised cells is considered next. Addition of Co²⁺ instantly exposed uphill Ca²⁺ extrusion by the PMCA. Co²⁺ blocks ionophore-mediated Ca²⁺ transport and is itself rapidly transported to equilibrium (Tiffert et al. 1984; Brown & Simonsen, 1985). Within red cells Co²⁺ acts as a low-affinity Mg²⁺ substitute for the Na⁺ and Ca²⁺ pumps (Richards, 1988; Raftos & Lew, 1995), with no detectable effect on the PMCA at physiological [Mg²⁺]_i levels. In parasitised cells the rapid build up of the internal Co²⁺ concentration would block ionophore-mediated Ca²⁺ transport between parasite and host. Thus, almost immediately after Co²⁺ addition, Ca²⁺ traffic to and from the red cell Ca²⁺ pool would be entirely dependent on endogenous active and passive Ca²⁺ transport across parasite and red cell plasma membranes. The parasitophorous vacuolar membrane which surrounds the parasite membrane is assumed to be devoid of active Ca²⁺ transport mechanisms and to represent no permeability barrier (Desai & Rosenberg, 1997). Active Ca²⁺ extrusion by a PMCA with normal V_max would be expected to rapidly empty the host cell Ca²⁺ pool (V_max measurement stage in Fig. 3), exposing residual Ca²⁺ trapped within parasite compartments. To a first approximation, this is the pattern observed in all the experiments of this series (Figs 2 and 3).

In suspensions of parasitised cells, pump V_max was marginally but systematically lower than in cohort or co-cultured cell suspensions (Fig. 3). It is doubtful whether this minor difference reflects a genuine pump inhibition in parasitised cells. Factors which could account for a minor apparent V_max inhibition are: Ca²⁺ redistribution between the rather limited Ca²⁺ pool of the host cell and that of the parasite, after Co²⁺ addition; the increased coefficient of variation resulting from the large residual Ca²⁺ pools; and the increased variability observed in repeated V_max estimates on the same suspension (9.08-11.7 mmol (10¹³ cells)⁻¹ h⁻¹ in the experiment of Fig. 3b). The conclusion from this analysis is that the performance of the Ca²⁺ pump in parasitised red cells is not significantly affected by P. falciparum parasites up to the late trophozoite stage. The documented conservation of PMCA function also removes any concerns about the estimates of host cell V_max arising from variable proportions of uninfected cells or from minor differences in parasite developmental stage as a result of imperfect synchronisation.

A final consideration on pump V_max concerns the comparison between results obtained with cell suspensions of different parasitaemia levels. In the experiment of Fig. 3a, the parasitaemia was 50 %. Pump V_max appeared similar in co-cultured and infected cells, and a superficial interpretation of this result may attribute the normal V_max to the 50 % cohort cells in the parasitised cell suspension, with minor or null contribution from the parasitised cells. Expression of mean cell Ca²⁺ content and flux rates per 10¹³ cells precludes that interpretation. If only 50 % of cells had contributed a normal V_max rate, the measured V_max would have been half-normal if expressed as millimoles per 10¹³ cells per hour. Thus, in the experiment of Fig. 3a, the similarity of V_max between co-cultured and parasitised cell suspensions suggests that pump performance was similar in the cohort and parasitised cells. That this is so, however, could only be confirmed at very high parasitaemia, as in the experiment of Fig. 3b, because the result in Fig. 3a may also reflect compensated differences between cohort and parasitised cells.

After the initial V_max, the residual Ca²⁺ retained within parasite compartments continued to be extruded from the cells, but at a much reduced rate. This extrusion was also uphill and therefore PMCA mediated. The emptying of the red cell Ca²⁺ pool during the V_max period changed the environment provided to the parasite, from a high-Ca²⁺ condition immediately after Co²⁺ addition, to its physiological low-Ca²⁺ condition. Residual Ca²⁺ traffic from parasite to host was therefore very probably down-gradient. After the initial V_max period, extrusion of residual Ca²⁺ settled to rates of between 0.6 and 1.2 mmol (10¹³ cells)⁻¹ h⁻¹ (Fig. 3). This presumed flow of Ca²⁺ from parasite to host may reflect properties of Ca²⁺ gradients and endogenous Ca²⁺ transport pathways at the parasite plasma membrane, amenable to further investigation.

A main additional factor which may influence the rate of residual Ca²⁺ efflux is the availability of ATP to the pump. The sudden ionophore-induced Ca²⁺ load fully activated a normal functioning PMCA which would have hydrolysed ATP with a Ca²⁺:ATP stoichiometry of 1:1 (Rega & Garrahan, 1986). Normal uninfected red cells lack the metabolic capacity to restore ATP at that rate, whatever the glycolytic substrate (McManus, 1967; Dagher & Lew, 1988). The combined operation of the PMCA ATPase, adenylate kinase and AMP deaminase enzymes leads to irreversible ATP depletion by conversion of AMP to IMP (inosine monophosphate) (Lew, 1971; Almaraz et al. 1988), processes which may, or may not, be conserved in cells with mature stage parasites. In normal red cells, ATP depletion becomes limiting to Ca²⁺ pump function only after about 20–30 min of pump activation, but in parasitised cells, with reduced host cell volume and ATP pool, ATP may become rate-limiting earlier. On the other hand, parasitised cells are known to have hugely increased glycolytic rates, with transport of ATP from parasite to host via a nucleotide translocase in the parasite plasma membrane (Kanaani & Ginsburg, 1989). The extra ATP consumption by the Ca²⁺-saturated PMCA represents a small fraction of the overall glycolytic rate of cells with mature parasites, but the relevant open question here is whether the rate of ATP translocation can match the rate of ATP consumption by the Ca²⁺-saturated pump of the host cell. It is worth noting that the residual Ca²⁺ pools generated by the Co²⁺-exposure protocol enable direct access to parasite Ca²⁺ pools with ⁴⁵Ca²⁺ of known specific activity, thus opening new ways of investigating the nature and dynamic behaviour of parasite Ca²⁺ pools.

This analysis suggests that the different experimental stages of the Co²⁺-exposure protocol can be refined to open new areas of investigation on the Ca²⁺ homeostasis of P. falciparum-infected cells, such as stage-related growth in parasite membrane area, cytoplasmic Ca²⁺ buffering within the parasite, the dynamics of parasite Ca²⁺ pools, effects of pharmacological agents on these functions, and study of the participation of Ca²⁺ in the mechanism of action of antimalarial drugs.