INCREASED
GLUCOSE PERMEABILITY IN BABESZA Z30VISINFECTED ERYTHROCYTES JOANNE M. UPSTON and ANNETTE N. CERO*
School of Biochemistry, University of New South Wales, P.O. Box I, Kensington, New South Wales 2033, Australia (Received 3 February 1989; accepted 6 August 1989) Abstract-UPsToN J. M. and GERO A. M. 1990. Increased glucose permeability in Bubesiu bovis-infected erythrocytes. International Journalfor Parusitology 20: 69-K Glucose influx into bovine erythrocytes was found to be signi~cant~y increased upon infection with the parasite, Bubesiu hovis. The inilux of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport. such as cytochalasin B and phloretin (0.1-100 PM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-p glucoside) at concentrations over the range of 10-500 PM. Further inhibition of glucose uptake by adenosine (2.515mM) was found to occur in B. bovis-infected bovine erythrocytes. suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.
INDEX KEY WORDS: Glucose influx; Babesiu bovis; glucose membrane erythrocytes; cytochalasin B; phioretin; phloridzin; adenoine.
INTRODUCTION
permeability;
bovine
However, relatively little is known of the mechanisms whereby an intraerythrocytic organism, such as a parasite, may induce host membrane alterations, or of the characteristics of such changes. Insights into membrane alteration, particularly derived from the study of Plasmodium infections, suggested that increased membrane permeabihty may be an important parasitic mechanism to obtain metabohtes not normally provided by the host erythrocyte and to discard waste products of intraerythrocytic metabolism. A general increase in the permeability of the parasitized erythrocyte membrane, while of obvious nutritional advantage to the parasite, may also be of use as a chemotherapeutic target, as compounds, not normally permeable and selectively toxic, may gain entry to the infected erythrocyte. The bovine erythrocyte is unusual in that it is virtually impermeable to glucose and nucleosides (Kim, 1983; Duhm, 1974). Erythrocytes and intraerythrocytic Babesia parasites, like Plasmodium, are unable to synthesize purine nucleosides de nova and, therefore, rely on obtaining these metabolites from the extracellular medium (Irwin, Young & Purnell, 1978; Conrad, 1986). In accordance with this obligate requirement, nucleoside transport has been found to be induced in bovine erythrocytes upon infection with B. bovis (Gero, 1989). In normal human erythrocytes, glucose and nucleosides cross the plasma membrane via facilitated diffusion utilizing specific transport proteins. It has been demonstrated that nucleosides can interact with the glucose transporter of human erythrocytes at the inner face of the membrane, suggesting structural similarities (Jarvis, 1988). The relationship of the two
THE anucleate red blood cell cannot provide all nutrients required by intraerythrocytic parasites for maturation. The entry of solutes into parasitized erythrocytes, recently studied in Plasmodium infections, has shown that pronounced changes occurred in the membrane of the infected erythrocyte, such that ~~eability to both electrolytes and non-electrolytes was significantly altered (Sherman, 1988). Despite the recognized similarities in Plasmodium and Babesia infections, particularly their pathological manifestations and intraerythrocytic development, little is known about the membrane permeability of Babesia-infected erythrocytes to essential nutrients. Early studies of Bubesia ~~~~a~~j-infect~ mouse erythrocytes demonstrated that r-glucose (an unnatural isomer), as well as o-glucose, could permeate the infected cell (Homewood, Neame & Momen, 1975). In addition, increased utilization of glucose had been shown to occur in B. rodhainiinfected rat erythrocytes (Rickard, 1969) and B. microti-infected mouse erythrocytes (Momen, 1979). However, the permeability of glucose in bovine erythrocytes infected with the economically important cattle strains of Babesia, such as B. bovis, has not been investigated. The human erythrocyte has been used extensively to study membrane transport systems and more recently to examine permeability changes caused by chemical modifications of the erythrocyte membrane and by pathological conditions (Ginsburg & Stein, 1987).
* To whom all correspondence should be addressed. 69
J. M. UPSTON and A. M. GERO
70
transporters has not been widely studied in other mammalian erythrocytes and rarely in parasitized erythrocytes. B. rodhaini-infected rat erythrocytes have been found to utilize glucose at an enhanced rate compared to the uninfected cell and addition of adenosine to the basal medium was demonstrated to greatly inhibit glucose uptake, apparently by competitive inhibition, as lactate production was unaltered (Barry, 1982). This study suggested that interactions occurred in glucose and nucleoside transport in B. rodhaini-infected cells and may arise from parasite-effected membrane perturbations resulting in alterations in permeability. The alteration in glucose uptake and/or interaction with the induced nucleoside transport in B. bovis-infected bovine erythrocytes, and a study of glucose influx properties into B. bovis-infected cells, are the subjects of the present investigation. MATERIALS
AND METHODS
Chemicals. 2-Deoxy-D[2,63H] glucose (1.44 TBq mmol~ ‘), [3H]H,0 (18.5 MBq mmol-‘) and [U-‘4C] sucrose (20.0 GBq mmol- ‘) were obtained from Amersham International, Amersham, Buckinghamshire, U.K. Cytochalasin B, phloretin, phloridzin (phloretin-2-/?-o-glucoside) and adenosine were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A. Stock solutions of cytochalasin B, phloretin and phloridzin were first prepared in absolute ethanol and then diluted in PBS (containing 0.15 M-NaCl, 1.86mM-KH,PO,, 4.8 mM-K2HP04, pH 7.4) so that the final concentration of ethanol was less than 0.5% v/v. Dow Corning Silicon oils were obtained from Ajax Chemicals, Blacktown, N.S.W., Australia. A silicon oil mix, of density 1.03 g ml-‘, was prepared by combining three different oil grades (parts by weight); 702 oil (45 centistokes (cs) viscosity, 80 parts), 200 oil (1.5 cs viscosity, 14 parts) and 200 oil (5 cs viscosity, 7 parts). 2,5-Diphenyloxalole (PPO), scintillation grade, was obtained from Koch Light Ltd, U.K. and CF-I 1 fibrous cellulose powder was obtained from Whatman BioSystems, Kent, U.K. All other chemicals were reagent grade commercial products. Deionized water was used in all preparative procedures. Preparation of erythrocyte suspensions. Virulent Babesia bovis (Samford strain) (Mahoney & Wright, 1976) was passaged through splenectomized calves and blood was collected on the day of experimentation into citrate phosphate dextrose. Leukocytes in blood samples normally settled in a layer above the red blood cells after centrifugation and were removed from erythrocyte suspensions by aspiration. However, in blood samples containing parasitized erythrocytes, leukocytes were found to be widely dispersed after washing and centrifugation and were subsequently removed by pouring the washed cells (10% hematocrit in PBS) through a dry CF- I I cellulose column (Richards & Williams, 1973). Uninfected erythrocytes in the blood sample were lysed in 10 x volume of hypotonic saline, at a concentration which rendered parasite-infected erythrocytes viable (Mahoney, 1967). The concentration of NaCl used in the differential lysis varied between blood samples (range of 0.375*.45% w/v) and was determined for each blood sample obtained. The lysate was removed after centrifugation at 3000 x g for 10 min. Parasitemias greater than 50% were obtained using this method. The pellet was washed three times in PBS and resuspended to a concentration of 2 x 1O*cell ml- ’ for use in uptake assays. The
concentration of all erythrocyte suspensions was enumerated using a model Zb Coulter Counter. Uninfected bovine blood (500 ml) was obtained from a normal, healthy cow and defibrinated using glass beads. The blood was washed in PBS and centrifuged at 3000 x gfor 5 min. The supernatant and white cell layer were removed by aspiration and the red cells resuspended in 10 x volume of PBS. The washing procedure was repeated a further two times, after which the cells were resuspended in an equal volume of Vega y Martinez (VYM) phosphate buffered saline solution composed of CaCl,. 2H,O (0.016 g), KC1 (0.400 g), KH, PO4 (1.415 g), MgS04. 7H,O (0.154 g). Nag HP04. 7H,O (1.450 g), NaCl (7.077 g) and glucose (20.5 g) in 1 1 of double distilled, deionized water containing 0.25 mM adenine and 0.50 mM guanosine (Vega, Buening, Green&Carson, 1985). Erythrocytes were stored at 4’C in this solution and remained viable for up to 2 weeks. Cells used in assays were removed from the storing solution, washed three times with PBS and finally diluted in PBS. Human type 0 erythrocytes were obtained from the New South Wales Red Cross Blood Transfusion Service and washed in PBS. After centrifugation at 3000 x gfor 5 min the supernatant and white cell layer were removed by aspiration. The red cells were washed a further two times in PBS, with removal of any remaining white cells between washings, and finally resuspended in PBS. Fresh, heparinized mouse erythrocytes were collected from Balb/C mice, washed twice in PBS and resuspended to a 10% hematocrit. Leukocytes were removed from the suspension by pouring through a CF1 I cellulose column, as described for B. bovis-infected erythrocytes. The erythrocytes were centrifuged at 3000 x g for 5 min and resuspended in PBS. Glucose uptake assays. Assays were initiated by mixing equal volumes of [3H]-labelled permeant and erythrocytes of concentration, 2 x lo* cell ml ‘, at 22°C. At specified times, between 0 and 30 min. 200 ,uI aliquots of this reaction mix were removed and layered over 150 ~1 silicon oil mix in 1.5 ml Eppendorf centrifuge tubes. The samples were immediately centrifuged at 16,000 x gfor a minimum of 15 s to pellet the erythrocytes below the oil layer. The density of the silicon oil mix (1.03g ml- ‘), used in all experiments, was such that the cells separated from the unused permeant upon centrifugation, thereby effectively stopping the reaction. Additionally, the oil mix prevented lysed cells and ‘free’ parasites from pelleting during centrifugation. Therefore, the radioactivity detected and used to calculate glucose influxes, represented only that taken up by intact erythrocytes. For zero time samples, 100 ~1 of [3H]-labelled permeant was layered over 150 ~1 silicon oil mix. One hundred microlitres of cell suspension was quickly added to the permeant and the tube immediately centrifuged. After centrifugation, the unused permeant layer was removed by aspiration and the pellet processed as described previously (Gero, Scott, O’Sullivan & Christopherson, 1989; Paterson, Harley & Cass, 1984). Radioactivity in each pellet was counted in scintillant (10 ml), containing, toluene: Triton X-100 2:l v/v + 0.5% w/v PPO. using a Packard Tri-Carb 300 Liquid Scintillation System. Inhibitors, diluted in PBS, were incorporated into the assay by mixing with the [3H]-labelled permeant prior to addition of cells. In determining the cell water space, [3H]H,0 and [U-14C] sucrose were incorporated into the assay in the place of the permeant. Glucose uptake measurements were carried out at 22’C, however, the physiological temperature of bovine species is approximately 37°C. Thus, glucose influx was also measured at 37°C to determine if an increase in temperature exhibited any significant effect in normal bovine erythrocytes. Glucose uptake into normal bovine erythrocytes exposed to
71
Glucose influx in B. bovis serllrn from +zcred calves. Normal bovine erythrocytes susnended in PBS at a conc~tration of 4 x IO*cell ml-‘, weie incubated at 37°C for 1h in serum obtained from a calf infected with B. bovis. The erythrocytes were washed three times in PBS and finally diluted in PBS to a concentration of 2 x 10’ cell ml-‘. Glucose uptake into the erythrocytes was assayed at 22°C as described above. Kinetics of glucose entry into parasitized erythrocytes.
Kinetic constants were calculated by computer analysis of data using a linear least-squares regression program based on that developed by Cleland (1979). For the kinetic experiments described, the time of exposure of erythrocytes to the permeant was limited (4 min) such that initial velocities of glucose influx were measured. Initial velocities of glucose influx were determined by extrapolation of the linear part of each curve, over the first 2-5 min, and measuring the amount of rH] glucose @moles) incorporated per ~1 erythrocytes per min. Cell size and determination of total water and extracellular volumes. The volume of the erythrocyte pellet was obtained from the calculation of space taken up by rH] H,O and [U‘“Cl sucrose. [‘HI H,O is djstributed evenly throughout the pellet and able to penetrate the erythrocytes and therefore represents the volume occupied by the cell pellet and any liquid trapped. For bovine erythrocytes the percentage mean water content is reported to be 7 1% (Rich, Sha’afi, Barton & Solomon, 1967). [U-“C] Sucrose, which cannot permeate the erythrocytes, was used to determine the extracellular volume of the pellet. Pellet volume (1) and the volume of individual cells (2) were calculated from the following formulae; VP = Vr/H - G-z
11)
where: VR= pellet volume; VT = total water space; H = 0.71 (fractional water content of bovine erythrocytes); VEcs = extracellular water space. vc = v&r
(2)
where: Vc = volume of cell; Cr = total number of cells in pellet. RESULTS Glucose in&x into normai and infected erythrocytes Glucose uptake into normal human, mouse and bovine erythrocytes and B. bovis-infected bovine erythrocytes was measured over 30 min at 22°C using a concentration of glucose (60 mw) which was saturating for the human and mouse cells (Fig. 1). The uptake of glucose into human and mouse erythrocytes was shown to increase with time and also found to be nonconcentrative. The intracellular concentration of glucose in both normal human and mouse red blood cells equilibrated with the extracellular glucose concentration within 30 min (Table 1). However, bovine erythrocytes displayed a very low permeability to glucose (Fig. 1) and at 30 min were found to have an intracellular concentration approximately l/IO of both human and mouse erythrocytes. An increase in the assay temperature to 37°C did not significantly increase glucose permeability into normal bovine erythrocytes (Table 1). However, infection of bovine erythrocytes with B. bavis substantially altered glucose uptake (Fig. I), as the intracellular concentration of glucose in a preparation of 53% infected erythrocytes
Time (min)
FIG. 1. Glucose uptake into normal human, mouse and bovine and B. bovis-infected bovine erythrocytes. Normal human (A), mouse (0) and bovine ( W) and B. bovis-infected bovine erythrocytes (53% parasitemia) (0) were suspended in PBS (2 x lo* cell ml-‘) at 22°C containing [3H]glucosc (60 mM). At the times shown, 200 ~1 was layered onto oil and cells pelleted by centrifugation. The [‘HI-content of the cell pellet was determined and expressed as nmole glucose/p1 cell water, which represented the permeant equivalent in the cell pellet with subtraction of background radiolabel attributable to extracellular water space. The values depicted represent triplicate experiments.
TABLEI-PROPERTIESOF GLIJCOSE UPTAKE INTONORMAL AND PARASITIZED ERYTHROCYTES Erythrocytes
Normal human Normal mouse Normal bovine Normal bovine (37”C)t B. bovtr-infected (53% parasitemia) Normal bovine + infected serum
Initial velocity Intracellular (nmole glucose concentration of pl cell H,o- ’ glucose fmM) min - ‘) at 22°C after 30 min 18.5 10.5 0.6 0.9 5.0
6.1 + 0.1 12.1 f 0.7 34.8 f 0.7
1.1
9.6 f: 0.6
56.0 f 0.3* 63.5 f 0.5
* The values represented are the mean f S.D.of three replicates. t Uptake of glucose (60 mM) into normal bovine erythrocytes was measured at both 22 and 37°C as described in the Methods section.
72
J.M.
UPST~N
GERO
andA.M.
was found to increase approximately six-fold over the uptake in uninfected bovine cells over a 30 min period (Table 1). The puri~~ation procedure used to isolate B. bovisinfected erythrocytes did not affect the permeability of the cells as the influx of metabolites was found to be proportional to the percentage of infected celIs in each preparation, irrespective of whether the cells had been subjected to the lysis step or not (Gero, 1989). E#ect qf serum from infected calves on glucose uptake into normal bovine eryihracyfes Normal bovine erythrocytes were incubated with serum from B. &v&infected calves. Under these conditions, a slight increase (less than two-fold) in glucose uptake into the normal bovine erythrocytes was observed (Table I). However this could be attributed to the rise in temperature of the experiment, as the erythrocytes exposed to infective serum were incubated at 37°C for 1 h and subsequently assayed at 22°C. Khetics ~~g~~cose entry into purasitized eryt~ir~cytes The kinetics of glucose influx into normal bovine erythrocytes could not be measured as the permeability of the cells to glucose was extremely low. Glucose influx in B. hovis-infected cells (53% parasitemia), using varying substrate concentrations, did not appear to saturate even at a concentration of glucose of 240 InM (Fig. 2), suggesting that glucose entered the parasitized bovine erythrocytes via simple diffusion.
, 60
120
180
240'
300
[glucose] (mM). FIG. 2. Effect of substrate concentration on rate of glucose influx into 8. hovis-infected bovine erythrocytes. The rate of [‘HIglucose influx (over 4 min) in a preparation of 53% B. bovis-infected bovine erythrocytes was assayed at 22’C as described in the Methods. with glucose concentrations ranging from IS to 240 mM. The values shown are the average of four replicate experiments.
loo-
80-
60-
40-
20-
Log [inhibitor] ($l) FIG. 3. Effect of inhibitors on glucose influx in B. bovis-infected bovine (53% parasitemia) and normal human erythrocytes. The effect of increasing concentrations (0.01-100 hi) of cytochalasin B and phloretin on uptake ofglucose (60 mM) in B. bovisinfected bovine erythrocytes (Fig. 3a 0, 0) and in normal human erythrocytes (5 mM) (Fig. 3b n , 0) over 4 min (B. 6oviF) or 1 min (human) intervals was determined as described in the Methods. Ceils were simultaneously exposed to both the inhibitor and the radiola~~led permeant prior to centrifugation through the oi1 layer. The values represent averages of triplicate ex~riments. Glucose uptake in the absence of any competing substrate was set at 100% activity. All other values were adjusted to this baseline.
13
GlucoseinfluxinB. bovis
Inhibition of glucose transport in parasitized erythrocytes To further characterize the properties of glucose
influx into 3. bovis-infected bovine erythrocytes, the effects of cytochalasin B, phloretin and phloridzin were investigated on a preparation of 53% parasitemia. Cytochalasin B was found to be without effect on the glucose influx into B. bovis-infected erythrocytes at concentrations of 0.1-100 PM (Fig. 3a). At the same range of concentrations, phloretin was found to inhibit glucose influx into B. bovis-infected erythrocytes by approximately 30% but was not shown to be concentration dependent. However, this ~on~ntration range of cytochalasin 3 and phloretin was sufficient to significantly inhibit glucose transport in normal human erythrocytes under the same experimentaf conditions (Fig. 3b). Conversely, phloridzin, over a concentration range of 10-500 ,UM, was demonstrated to significantly inhibit glucose uptake into B. bovisinfected bovine erythrocytes, whereas these concentrations did not affect glucose uptake into normal human cells (Fig. 4). relationship of glucose and nucleoside parasitized erythrocytes
tramport
in
The possible relationship of nucleoside and glucose transport in B. bovis-infected bovine erythrocytes was investigated using adenosine as an inhibitor of glucose uptake. Influx of glucose (60 mM) in B. bovis-infected bovine erythrocytes was found to be inhibited by
100
80
60
40
T
TABLET-PERCENT HUMAN AND B.
lNHIBITIONOFGLUCOSEINFLUXINTONORMAL bOYiS-INFECTED BOWNE ERYTHROCYTES BY ADENOSINE*
Normal human erythrocytes Adenosine % Inhibition (mM) 2 10 17
B. ~~v~-inf~~d bovine erythrocytes Adenosine (mM)
% Inhibition
2.5 10 15
62 -f: 6 57 f 6 57 f 3
0 0 7f2
* The influx of glucose into normal human erythrocytes was measured using 5 mM glucose. For B. bo~j~-infected bovine erythrocytes, the concentration of glucose was 60 mM. The values represented are the mean i S.D.of three replicates.
adenosine at concentrations between 2.5 and 15 mM (Table 2). A similar concentration range of adenosine (2-17 mM) had no effect on glucose uptake into normal human erythrocytes although the concentration of glucose used in the assay (5 IIIM)was significantly less than that used for B. bovis-infected cells (Table 2). Estimation
of cellular volumes
The cellular volumes of parasitized and uninfected bovine erythrocytes in the pellets beneath the oil in the assays were calculated from cell counts and intracellular volumes (Table 3). The presence of the Babesia parasite was not found to induce any change in the volume of the bovine erythrocyte, in contrast to the maIaria1 parasite in human erythrocytes which increased the cell volume (Gero, A. M. & Upston, J. M., unpublished data). The enhancement in B. bovisinfected erythrocyte membrane permeability, therefore, did not appear to be due to an alteration of host cell volume. TABLE
~-CELLULAR
VOLUMES OF PARASITIZEDAND NORMAL BOVINE~R~T~R~~T~S
Volume (fl)
Cell type
Normal bovine erythrocytes B. bovis-infected bovine erythrocytes?
20
37.9 f 2.5 (7)* 37.2 f 2.3 (3)
* The values in parentheses refer to the number of determinations used to calculate the average cellular volume l S.D (in A, lo-” X iitre). t Values represented correspond to parasitemias of 90%.
E -2
-3
0
1
2
3
DISCUSSION Log [Phloridzin} (PM). FIG. 4. Effect of phloridzin on glucose uptake in B. bovisinfected bovine and normal human erythrocytes. The effect
of phloridzin (0.01-I mM) on glucose (60 mm) uptake (over 4 min) in B. bovis-infected bovine erythrocytes (A) and normal human erythrocytes (a) was assayed as described for Fig. 3. Again, the values represent three replicate experiments.
Bovine erythrocytes, which are virtually impermeable to glucose, were shown to display dramatic increases in glucose permeability following infection with B. bovis. The results of kinetic analyses appeared to provide evidence for a non-saturable mode of glucose ‘transport’ as there was no departure from linearity even at concentrations of glucose 70-fold higher than
74
J. M. UPSTONand A. M. GERO
normal bovine plasma levels (3.5 RIM) (Fig. 2). However, not all criteria required for a simple diffusion model were satisfied. Simple diffusion is defined by the following: it is dependent on the maintenance of a concentration gradient, net movement is from a region of high concentration to a lower one, there is a low temperature dependence, the process is not saturable and there is no competition for substrate by structurally related molecules (She~an, 1988). Glucose uptake into parasitized bovine cells was found to be non-saturable and was not affected by increasing concentrations of established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin, at the concentrations shown to be effective in human erythrocytes (Fig. 3). Although phloretin reduced glucose influx into B. b&s-infected cells to approximately 70% of the control, the inhibition was not concentration-dependent over the concentration range tested. These data suggest that glucose permeability in B. bovis-infected erythrocytes is different, with respect to cytochalasin B and phloretin inhibition, from the normal human red cell glucose transport. Glucose influx, in B. bovis-infected cells was, however, significantly inhibited by phloridzin and adenosine, suggesting deviation from the simple diffusion model. In comparison, the transport of the amino acid, Lgiutamine, into the normal human erythrocyte has also been reported to be saturable, whilst t-glutamine influx into Plasmodium fulciparum-infected human erythrocytes was found to be a non-saturable process (Elford, Haynes, Chulay & Wilson, 1985; Ellory, Elford & Newbold, 1988). In addition the nonsaturable transport of L-glutamine was demonstrated to be specifically inhibited by piperine, artemetin and artesunate, whereas the uninfected erythrocyte transport remained unaffected. These results appear to be characteristically similar to those obtained for glucose influx into B. b&s-infected erythrocytes. suggesting that the mechanisms whereby Plasmodium and Babesia modify the membrane of their host cells to allow nutrient permeability may be analogous. Further, our data suggest that the component of glucose uptake found in B. b&s-infected bovine erythrocytes behaves differently to that found in normal mammalian cells. Phloridzin, at concentrations greater than 10 PM, effectively inhibited glucose influx into the parasitized cells, whilst the same concentration range of phloridzin did not inhibit glucose influx into normal human erythrocytes (Fig. 4). Phloridzin has been demonstrated to have antibabesial activity in in vitro cultures of B. bovis, with a IDSo (50% inhibitory dose) of 50 puw (Nott, Gero, O’Sullivan & Bagnara, unpublished data). Kutner, Breuer, Ginsburg & Cabantchik (1987) have also demonstrated that phloridzin effectively inhibits intraerythrocytic malarial growth in in vitro cultures of P. falciparum and have suggested that the antimalarial action may be associated in part with inhibition of solute transfer through membrane alterations induced
in the host cell membrane by the intracellular parasite, or via phloridzin acting directly with a parasite constituent. Our data with phloridzin again suggest that both intraerythrocytic parasites induce host cell membrane alterations to allow increased permeability. It could be envisaged that permeability changes in the membrane of parasite-infected erythrocytes proceeded through utilization of specific transport sites which were present, but not functionally active in the uninfected cell, such as reutilization of foetal calf red cell transporters. However, no glucose transport into newborn calf erythrocytes has been demonstrated (Kim, 1983) diminishing the possibility of the parasite activating foetal transport sites in the host cell to increase permeability. In addition, uninfected bovine cells did not increase in their permeability to glucose upon exposure to serum from infected calves, thereby suggesting that serum components were not responsible for the increased permeability of the B. bovisinfected cells to glucose. Adenosine was found to inhibit glucose influx in B. bovis-infected bovine erythrocytes suggesting an interaction of adenosine with the new or altered component of glucose ‘transport’ in these cells (Table 2). Similarly in B. rodhaini-infected rat erythrocytes, adenosine was found to competitively inhibit glucose uptake (Barry, 1982). In normal human erythrocytes both the glucose and nucleoside transport proteins migrate as a complex on sodium dodecyl sulphate polyacrylamide gel electrophoresis (band 4.5) and were found to have an apparent molecular weight of 4.5,00@-65,000. Despite the fact that the two proteins have not yet been separated, they could be discriminated on the basis of selective inhibitor binding (Paterson & Cass, 1985; Wheeler & Hinkle, 1985) and recognition by monoclonal antibodies (Tai & CarterSu, 1988). A recent study of the two proteins has revealed that nucleosides can interact with the glucose transporter of human erythrocytes (Jarvis, 1988). However, the glucose and nucleoside transporters are not simultaneously active in all mammalian species. For example, adult pig erythrocytes are impermeable to sugars, but retain functional nucleoside transport (Young, Paterson & Henderson, 1985). In comparison, there is no nucleoside transport into the bovine erythrocyte and, as demonstrated above, although the bovine erythrocyte glucose transport protein is functional, it transports this sugar at a rate of approximately l/2000 that of human erythrocytes (Hoos, Tarpley & Regen, 1972). The dramatic increase in glucose influx, which we have demonstrated in B. bovis-infected bovine erythrocytes, was found to be accompanied by induction of nucleoside transport (Gero 1989) and thus the possible reIationship of glucose and nucleoside transport in Lf. bovis-infected bovine cells was explored. The data in Table 2 indicate a possible relationship between adenosine and glucose transport, as adenosine (2.5-l 5 mM) appeared to inhibit glucose influx in B. bovis-infected cells. Three concentrations
Glucose
15
influx in B. bovis
of adenosine within a small range were chosen but an ID,, such as in Fig. 3 was not attempted. Further work on this phenomenon will be investigated but is not within the scope of this paper. Although adenosine was found to inhibit glucose transport in B. bovisinfected bovine erythrocytes (Table 2), the converse did not occur, as glucose (at concentrations up to 120 mM) was demonstrated to be without effect on adenosine transport (1 PM) (Gero, A. M., unpublished). Our results suggest, however, that adenosine interacts with the new or altered component of glucose ‘transport’ in B. bovis-infected bovine erythrocytes. Invasion of bovine erythrocytes with B. bovis did not appear to induce any change in the volume of the infected cell (Table 3). In comparison, the presence of mature intraerythrocytic forms of P. falciparum (trophozoites and schizonts) has been shown to increase the volume of infected human cells (Gero, A. M. & Upston, J. M., unpublished data). It is not known whether an increase in the volume of parasitized erythrocytes, due to changes in the membrane structure, could, therefore, account for alterations in membrane permeability in Plasmodium infections. However, the absence of an alteration in the bovine erythrocyte volume upon infection with B. bovis suggested that other mechanisms of increasing membrane permeability may operate in Babesiu infections. An alternative suggestion, to explain the mechanism whereby glucose (and other nutrients) permeability is altered by invasion of the bovine erythrocyte by B. bovis, may be misadjustment between phospholipid and protein in the erythrocyte membrane which has been used to account for the altered membrane properties of Plasmodium-infected erythrocytes (Schwartz, Olson, Raventos-Suarez, Yee, Heath, Lubin & Nagel, 1987). Increased permeability of parasitized cells could arise if parasite encoded proteins, embedded in the host cell membrane, did not form tight seals with the hydrocarbon chain of the phospholipids in the membrane (Ginsburg & Stein, 1987). Permeability changes apparent in Bubesia infections are similarly thought to arise through alterations in the phospholipid organization of the host cell membrane (Wright, Goodger & Clark, 1988) as B. bovis-infected bovine erythrocyte membranes have been found to contain parasite proteins (James, Montenegro-James, Fajfar-Whetstone, Montealegre, Erickson & Ristic, 1987). Additionally, B. bovisinfected bovine erythrocytes selectively bind to Heparin-Sepharose columns (Goodger, Mahoney & Wright, 1983), again suggesting that the membranes of the infected cells contain altered components. In conclusion, we have provided evidence that dramatic increases in glucose permeability occur in bovine erythrocytes upon infection with B. bovis. These data together with previous studies on rodent babesias (Rickard, 1969, Homewood et al., 1975; Momen, 1979; Barry, 1982), suggest that the babesia
parasite is able to alter the membrane permeability of the host cell to increase glucose uptake for its metabolic needs. The ‘transport’ mechanism whereby glucose enters B. bovis-infected erythrocytes appears to be characteristically different from that normally found in uninfected bovine and human erythrocytes. Also, adenosine was found to inhibit glucose influx in B. bovis-infected cells suggesting an interaction of the new or altered component of glucose ‘transport’ and the induced nucleoside transport. Further work in this area would elucidate whether the new or altered ‘transport’ of glucose is a leak, pore or an altered transporter in the parasitized erythrocyte. In addition, characterization of glucose (and other nutrients) influx mechanisms in infected cells and viable parasites, freed from the host cell membrane, may help to determine modes whereby parasite derived proteins are inserted into the membrane of the infected cell and whether changes in permeability occur due to alterations in the membrane structure. Acknowledgements-We wish to thank Dr I. G. Wright, Division of Tropical and Animal Production, CSIRO, Qld, for provision of Babesia bovis-infected erythrocytes and serum from infected calves and W. J. O’Sullivan for his interest. This work was supported by a grant to AMG from the National Health and Medical Research Council of Australia and the UNDP/World Bank/WHO Special Programme for the Research and Training in Tropical Diseases.
REFERENCES BARRY D. N. 1982. Metabolism of Babesia parasites in vitro. Glucose and energy metabolism and survival of Babesia rodhaini in a basal medium with and without adenosine. Australian Journal of E.uperimental Biology and Medical Science 60: 159-165. CLELAND W. W. 1979. Statistical analysis of enzyme kinetic data. Methods in Enzymology 63A: 103-138. CONRADP. A. 1986. Uptake of triated nucleic acid precursors by Babesia bovis in vitro. International Journal for Parasitology 16: 263-268. DUHM J. 1974. Inosine permeability and purine nucleoside phosphorylase activity as limiting factors for the synthesis of 2, 3-bisphosphoglycerate from inosine, pyruvate and inorganic phosphate in erythrocytes of various mammalian species. Biochimica et Biophysics Acta 343: 89-100. ELFORD B. C., HAYNESJ. D., CHULAYJ. D. % WILSON R. J. M. 1985. Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum. Molecular and Biochemical Parasitology 16: 43-60. ELLORYJ. C., ELFORD B. C. &NEWBOLD C. I. 1988. Transport mechanisms across cell membranes. Parasitology 96: s5s9. GERO A. M. 1989. Induction ofnucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes. Molecular and Biochemical Parasitology 35: 269~276. GERO A. M., SCOTT H. V., O’SULLIVAN W. J. & CHRISTOPHERSONR. I. 1989. Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites. Molecular and Biochemical Parasitology 34: 87-98.
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J. M. UPSTONand A. M. GERO
H. & STEIN W. D. 1987. Biophysical analysis of novel transport pathways induced in red blood cell membranes. Journal of Membrane Biology 96: I-10. GRUDGERB. V., MAHONEYD. F. & WRIGHTI. G. 1983. Babesia bovis: attachment of infected erythrocytes to Heparin-Sepharose columns. Journal of Parasitology 69:
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248-249.
HOMEWARD C. A., NEAME K. D. & MOMEN H. t975. Permeability of erythrocytes from mice infected with Babesia rodhajni. Annals Parasiro1og.v 69: 429-434.
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Hoes R. T., TARPLEVH. L. & REGEND. M. 1972. Sugar transport in beef erythrocytes. Biochimica et Biophysics Acta 266: 174181. IRWINA. D., YOUNG E. R. & PURNELL R. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. in cattle and mice. International Journalfor Parasitology 8:
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PATERSONA. R. P. & CASS C. E. 1985. Transport of nucleoside drugs in animal cells. In: Infernarional Encyclopedia ofPharmacology and Therapeutics (Edited by GOLDMAN J. D.), pp. 309-329. Pergamon Press, Oxford. RICH G. T., SHA.AFIR. I., BARTON T. C. & SOLOMON A. K. 1967. Pe~eability studies on red cell membranes of dog, cat and beef. Journalof General Physiology 50: 2391-2405. RICHARDS W. H. G. &WILLIAMSS. G. 1973. The removal of leukocytes from malaria infected blood. Annals of Tropical Medicine and Parasitology 67: 249-250.
RICKARDM. D. 1969. Carbohydrate metabolism in Babesia rodhaini: differences in the metabolism of normal and infected rat erythrocytes. Experimental Parasitology 25: 16-31.
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JAMESM. A., MONTE~EGRO-JAMES S., FAJFAR-WHETSTONE C., MONTEALECRE F., ERICKSON J. & RISTICM. 1987. Antigenic relationship between Plasmodium fal~iparum and Babesia bovis: reactivity with antibodies to culture-derived soluble exoantigens. Journal of Protozoology 34: 328-332. JARVISS. M. 1988. Inhibition bv nucleosides of alucose transport activity in human e>ythrocytes. Biochemical Journal 249: 383-389. KIM
H. D. 1983. Post-natal changes in energy metabolism of mammalian red blood cells. In: Red Blood Cells of L3omestic Animals (Edited by AGARN. S. & BOARDP. G.), pp. 339-355. Elsevier Science, The Netherlands. KUTNER S., BREUERW. V., GINSBURG H. & CABANT~HIK Z. I. 1987. On the mode of action of phlorizin as an antimalarial agent in in vivo cultures of Plasmodium falciparum. Biochemical Pharmacology 36: 123-l 29. MAHONEYD. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Experimental Parasitology 20: 232-24
PATERSON A. R. P., HARLEYE. R. & CASSC. E. 1984. Inward fluxes of adenosine in erythrocytes and cultured ceils measured by a quenched-flow method. Biochemical Journal
1.
MAHONEYD. F. & WRIGHTI. G. 1976. Babesia argentina: immunization of cattle with a killed antigen against infection with a heterologous strain. ~e~~r~nar~ Parasjtology 2: 2733282.
MOMENH. 1979. Biochemistry of intraerythrocytic parasites II: comparative studies in carbohydrate metabolism. Annals of Tropical Medicine and Parasitology 13:
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R. S., OLSON J. A., RAVENTOS-SUAREZ C., YEEM., HEATHR. H., LUBINB. & NAGELP. L. 1987. Altered plasma membrane phospholipid organisation in PZasmodiam filciparum-infected human erythrocytes. Blood 69: 401-
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SHERMAN I. W. 1988. Mechanisms of molecular trafficking in malaria. Parasitology 96: s57-s8 1. TAI P-K. K. & CARTER-SU C. 1988. Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters. Biochemistry 27: 6062607 1. VEGA C. A., BUENING G. M., GREENT. J. & CARSOHC. A. 1985. In vitro cultivation of Babesia bigemina. American Journal of Veterinary Research 46: 4 16420.
WHEELER T. J. & HINKLEP. C. 1985. The glucose transporter of mammalian cells. Annual Reviews in Physioiogy 47: 503517.
WRIGHTI. G., GRUDGERB. V. & CLARKEI. A. 1988. Immunopathophysiology of Babe.& bovis and Plasmod&m falciparum infections Parasitology Today 4: 2 i 4218. YOUNG
J. D., PATERSONA. R. P. & HENDERSON J. F. 1985. Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate. Biochimica eI Biophysics Arta 842: 2 14-224.
GLUCOSE PERMEABILITY IN BABESZA Z30VISINFECTED ERYTHROCYTES JOANNE M. UPSTON and ANNETTE N. CERO*
School of Biochemistry, University of New South Wales, P.O. Box I, Kensington, New South Wales 2033, Australia (Received 3 February 1989; accepted 6 August 1989) Abstract-UPsToN J. M. and GERO A. M. 1990. Increased glucose permeability in Bubesiu bovis-infected erythrocytes. International Journalfor Parusitology 20: 69-K Glucose influx into bovine erythrocytes was found to be signi~cant~y increased upon infection with the parasite, Bubesiu hovis. The inilux of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport. such as cytochalasin B and phloretin (0.1-100 PM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-p glucoside) at concentrations over the range of 10-500 PM. Further inhibition of glucose uptake by adenosine (2.515mM) was found to occur in B. bovis-infected bovine erythrocytes. suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.
INDEX KEY WORDS: Glucose influx; Babesiu bovis; glucose membrane erythrocytes; cytochalasin B; phioretin; phloridzin; adenoine.
INTRODUCTION
permeability;
bovine
However, relatively little is known of the mechanisms whereby an intraerythrocytic organism, such as a parasite, may induce host membrane alterations, or of the characteristics of such changes. Insights into membrane alteration, particularly derived from the study of Plasmodium infections, suggested that increased membrane permeabihty may be an important parasitic mechanism to obtain metabohtes not normally provided by the host erythrocyte and to discard waste products of intraerythrocytic metabolism. A general increase in the permeability of the parasitized erythrocyte membrane, while of obvious nutritional advantage to the parasite, may also be of use as a chemotherapeutic target, as compounds, not normally permeable and selectively toxic, may gain entry to the infected erythrocyte. The bovine erythrocyte is unusual in that it is virtually impermeable to glucose and nucleosides (Kim, 1983; Duhm, 1974). Erythrocytes and intraerythrocytic Babesia parasites, like Plasmodium, are unable to synthesize purine nucleosides de nova and, therefore, rely on obtaining these metabolites from the extracellular medium (Irwin, Young & Purnell, 1978; Conrad, 1986). In accordance with this obligate requirement, nucleoside transport has been found to be induced in bovine erythrocytes upon infection with B. bovis (Gero, 1989). In normal human erythrocytes, glucose and nucleosides cross the plasma membrane via facilitated diffusion utilizing specific transport proteins. It has been demonstrated that nucleosides can interact with the glucose transporter of human erythrocytes at the inner face of the membrane, suggesting structural similarities (Jarvis, 1988). The relationship of the two
THE anucleate red blood cell cannot provide all nutrients required by intraerythrocytic parasites for maturation. The entry of solutes into parasitized erythrocytes, recently studied in Plasmodium infections, has shown that pronounced changes occurred in the membrane of the infected erythrocyte, such that ~~eability to both electrolytes and non-electrolytes was significantly altered (Sherman, 1988). Despite the recognized similarities in Plasmodium and Babesia infections, particularly their pathological manifestations and intraerythrocytic development, little is known about the membrane permeability of Babesia-infected erythrocytes to essential nutrients. Early studies of Bubesia ~~~~a~~j-infect~ mouse erythrocytes demonstrated that r-glucose (an unnatural isomer), as well as o-glucose, could permeate the infected cell (Homewood, Neame & Momen, 1975). In addition, increased utilization of glucose had been shown to occur in B. rodhainiinfected rat erythrocytes (Rickard, 1969) and B. microti-infected mouse erythrocytes (Momen, 1979). However, the permeability of glucose in bovine erythrocytes infected with the economically important cattle strains of Babesia, such as B. bovis, has not been investigated. The human erythrocyte has been used extensively to study membrane transport systems and more recently to examine permeability changes caused by chemical modifications of the erythrocyte membrane and by pathological conditions (Ginsburg & Stein, 1987).
* To whom all correspondence should be addressed. 69
J. M. UPSTON and A. M. GERO
70
transporters has not been widely studied in other mammalian erythrocytes and rarely in parasitized erythrocytes. B. rodhaini-infected rat erythrocytes have been found to utilize glucose at an enhanced rate compared to the uninfected cell and addition of adenosine to the basal medium was demonstrated to greatly inhibit glucose uptake, apparently by competitive inhibition, as lactate production was unaltered (Barry, 1982). This study suggested that interactions occurred in glucose and nucleoside transport in B. rodhaini-infected cells and may arise from parasite-effected membrane perturbations resulting in alterations in permeability. The alteration in glucose uptake and/or interaction with the induced nucleoside transport in B. bovis-infected bovine erythrocytes, and a study of glucose influx properties into B. bovis-infected cells, are the subjects of the present investigation. MATERIALS
AND METHODS
Chemicals. 2-Deoxy-D[2,63H] glucose (1.44 TBq mmol~ ‘), [3H]H,0 (18.5 MBq mmol-‘) and [U-‘4C] sucrose (20.0 GBq mmol- ‘) were obtained from Amersham International, Amersham, Buckinghamshire, U.K. Cytochalasin B, phloretin, phloridzin (phloretin-2-/?-o-glucoside) and adenosine were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A. Stock solutions of cytochalasin B, phloretin and phloridzin were first prepared in absolute ethanol and then diluted in PBS (containing 0.15 M-NaCl, 1.86mM-KH,PO,, 4.8 mM-K2HP04, pH 7.4) so that the final concentration of ethanol was less than 0.5% v/v. Dow Corning Silicon oils were obtained from Ajax Chemicals, Blacktown, N.S.W., Australia. A silicon oil mix, of density 1.03 g ml-‘, was prepared by combining three different oil grades (parts by weight); 702 oil (45 centistokes (cs) viscosity, 80 parts), 200 oil (1.5 cs viscosity, 14 parts) and 200 oil (5 cs viscosity, 7 parts). 2,5-Diphenyloxalole (PPO), scintillation grade, was obtained from Koch Light Ltd, U.K. and CF-I 1 fibrous cellulose powder was obtained from Whatman BioSystems, Kent, U.K. All other chemicals were reagent grade commercial products. Deionized water was used in all preparative procedures. Preparation of erythrocyte suspensions. Virulent Babesia bovis (Samford strain) (Mahoney & Wright, 1976) was passaged through splenectomized calves and blood was collected on the day of experimentation into citrate phosphate dextrose. Leukocytes in blood samples normally settled in a layer above the red blood cells after centrifugation and were removed from erythrocyte suspensions by aspiration. However, in blood samples containing parasitized erythrocytes, leukocytes were found to be widely dispersed after washing and centrifugation and were subsequently removed by pouring the washed cells (10% hematocrit in PBS) through a dry CF- I I cellulose column (Richards & Williams, 1973). Uninfected erythrocytes in the blood sample were lysed in 10 x volume of hypotonic saline, at a concentration which rendered parasite-infected erythrocytes viable (Mahoney, 1967). The concentration of NaCl used in the differential lysis varied between blood samples (range of 0.375*.45% w/v) and was determined for each blood sample obtained. The lysate was removed after centrifugation at 3000 x g for 10 min. Parasitemias greater than 50% were obtained using this method. The pellet was washed three times in PBS and resuspended to a concentration of 2 x 1O*cell ml- ’ for use in uptake assays. The
concentration of all erythrocyte suspensions was enumerated using a model Zb Coulter Counter. Uninfected bovine blood (500 ml) was obtained from a normal, healthy cow and defibrinated using glass beads. The blood was washed in PBS and centrifuged at 3000 x gfor 5 min. The supernatant and white cell layer were removed by aspiration and the red cells resuspended in 10 x volume of PBS. The washing procedure was repeated a further two times, after which the cells were resuspended in an equal volume of Vega y Martinez (VYM) phosphate buffered saline solution composed of CaCl,. 2H,O (0.016 g), KC1 (0.400 g), KH, PO4 (1.415 g), MgS04. 7H,O (0.154 g). Nag HP04. 7H,O (1.450 g), NaCl (7.077 g) and glucose (20.5 g) in 1 1 of double distilled, deionized water containing 0.25 mM adenine and 0.50 mM guanosine (Vega, Buening, Green&Carson, 1985). Erythrocytes were stored at 4’C in this solution and remained viable for up to 2 weeks. Cells used in assays were removed from the storing solution, washed three times with PBS and finally diluted in PBS. Human type 0 erythrocytes were obtained from the New South Wales Red Cross Blood Transfusion Service and washed in PBS. After centrifugation at 3000 x gfor 5 min the supernatant and white cell layer were removed by aspiration. The red cells were washed a further two times in PBS, with removal of any remaining white cells between washings, and finally resuspended in PBS. Fresh, heparinized mouse erythrocytes were collected from Balb/C mice, washed twice in PBS and resuspended to a 10% hematocrit. Leukocytes were removed from the suspension by pouring through a CF1 I cellulose column, as described for B. bovis-infected erythrocytes. The erythrocytes were centrifuged at 3000 x g for 5 min and resuspended in PBS. Glucose uptake assays. Assays were initiated by mixing equal volumes of [3H]-labelled permeant and erythrocytes of concentration, 2 x lo* cell ml ‘, at 22°C. At specified times, between 0 and 30 min. 200 ,uI aliquots of this reaction mix were removed and layered over 150 ~1 silicon oil mix in 1.5 ml Eppendorf centrifuge tubes. The samples were immediately centrifuged at 16,000 x gfor a minimum of 15 s to pellet the erythrocytes below the oil layer. The density of the silicon oil mix (1.03g ml- ‘), used in all experiments, was such that the cells separated from the unused permeant upon centrifugation, thereby effectively stopping the reaction. Additionally, the oil mix prevented lysed cells and ‘free’ parasites from pelleting during centrifugation. Therefore, the radioactivity detected and used to calculate glucose influxes, represented only that taken up by intact erythrocytes. For zero time samples, 100 ~1 of [3H]-labelled permeant was layered over 150 ~1 silicon oil mix. One hundred microlitres of cell suspension was quickly added to the permeant and the tube immediately centrifuged. After centrifugation, the unused permeant layer was removed by aspiration and the pellet processed as described previously (Gero, Scott, O’Sullivan & Christopherson, 1989; Paterson, Harley & Cass, 1984). Radioactivity in each pellet was counted in scintillant (10 ml), containing, toluene: Triton X-100 2:l v/v + 0.5% w/v PPO. using a Packard Tri-Carb 300 Liquid Scintillation System. Inhibitors, diluted in PBS, were incorporated into the assay by mixing with the [3H]-labelled permeant prior to addition of cells. In determining the cell water space, [3H]H,0 and [U-14C] sucrose were incorporated into the assay in the place of the permeant. Glucose uptake measurements were carried out at 22’C, however, the physiological temperature of bovine species is approximately 37°C. Thus, glucose influx was also measured at 37°C to determine if an increase in temperature exhibited any significant effect in normal bovine erythrocytes. Glucose uptake into normal bovine erythrocytes exposed to
71
Glucose influx in B. bovis serllrn from +zcred calves. Normal bovine erythrocytes susnended in PBS at a conc~tration of 4 x IO*cell ml-‘, weie incubated at 37°C for 1h in serum obtained from a calf infected with B. bovis. The erythrocytes were washed three times in PBS and finally diluted in PBS to a concentration of 2 x 10’ cell ml-‘. Glucose uptake into the erythrocytes was assayed at 22°C as described above. Kinetics of glucose entry into parasitized erythrocytes.
Kinetic constants were calculated by computer analysis of data using a linear least-squares regression program based on that developed by Cleland (1979). For the kinetic experiments described, the time of exposure of erythrocytes to the permeant was limited (4 min) such that initial velocities of glucose influx were measured. Initial velocities of glucose influx were determined by extrapolation of the linear part of each curve, over the first 2-5 min, and measuring the amount of rH] glucose @moles) incorporated per ~1 erythrocytes per min. Cell size and determination of total water and extracellular volumes. The volume of the erythrocyte pellet was obtained from the calculation of space taken up by rH] H,O and [U‘“Cl sucrose. [‘HI H,O is djstributed evenly throughout the pellet and able to penetrate the erythrocytes and therefore represents the volume occupied by the cell pellet and any liquid trapped. For bovine erythrocytes the percentage mean water content is reported to be 7 1% (Rich, Sha’afi, Barton & Solomon, 1967). [U-“C] Sucrose, which cannot permeate the erythrocytes, was used to determine the extracellular volume of the pellet. Pellet volume (1) and the volume of individual cells (2) were calculated from the following formulae; VP = Vr/H - G-z
11)
where: VR= pellet volume; VT = total water space; H = 0.71 (fractional water content of bovine erythrocytes); VEcs = extracellular water space. vc = v&r
(2)
where: Vc = volume of cell; Cr = total number of cells in pellet. RESULTS Glucose in&x into normai and infected erythrocytes Glucose uptake into normal human, mouse and bovine erythrocytes and B. bovis-infected bovine erythrocytes was measured over 30 min at 22°C using a concentration of glucose (60 mw) which was saturating for the human and mouse cells (Fig. 1). The uptake of glucose into human and mouse erythrocytes was shown to increase with time and also found to be nonconcentrative. The intracellular concentration of glucose in both normal human and mouse red blood cells equilibrated with the extracellular glucose concentration within 30 min (Table 1). However, bovine erythrocytes displayed a very low permeability to glucose (Fig. 1) and at 30 min were found to have an intracellular concentration approximately l/IO of both human and mouse erythrocytes. An increase in the assay temperature to 37°C did not significantly increase glucose permeability into normal bovine erythrocytes (Table 1). However, infection of bovine erythrocytes with B. bavis substantially altered glucose uptake (Fig. I), as the intracellular concentration of glucose in a preparation of 53% infected erythrocytes
Time (min)
FIG. 1. Glucose uptake into normal human, mouse and bovine and B. bovis-infected bovine erythrocytes. Normal human (A), mouse (0) and bovine ( W) and B. bovis-infected bovine erythrocytes (53% parasitemia) (0) were suspended in PBS (2 x lo* cell ml-‘) at 22°C containing [3H]glucosc (60 mM). At the times shown, 200 ~1 was layered onto oil and cells pelleted by centrifugation. The [‘HI-content of the cell pellet was determined and expressed as nmole glucose/p1 cell water, which represented the permeant equivalent in the cell pellet with subtraction of background radiolabel attributable to extracellular water space. The values depicted represent triplicate experiments.
TABLEI-PROPERTIESOF GLIJCOSE UPTAKE INTONORMAL AND PARASITIZED ERYTHROCYTES Erythrocytes
Normal human Normal mouse Normal bovine Normal bovine (37”C)t B. bovtr-infected (53% parasitemia) Normal bovine + infected serum
Initial velocity Intracellular (nmole glucose concentration of pl cell H,o- ’ glucose fmM) min - ‘) at 22°C after 30 min 18.5 10.5 0.6 0.9 5.0
6.1 + 0.1 12.1 f 0.7 34.8 f 0.7
1.1
9.6 f: 0.6
56.0 f 0.3* 63.5 f 0.5
* The values represented are the mean f S.D.of three replicates. t Uptake of glucose (60 mM) into normal bovine erythrocytes was measured at both 22 and 37°C as described in the Methods section.
72
J.M.
UPST~N
GERO
andA.M.
was found to increase approximately six-fold over the uptake in uninfected bovine cells over a 30 min period (Table 1). The puri~~ation procedure used to isolate B. bovisinfected erythrocytes did not affect the permeability of the cells as the influx of metabolites was found to be proportional to the percentage of infected celIs in each preparation, irrespective of whether the cells had been subjected to the lysis step or not (Gero, 1989). E#ect qf serum from infected calves on glucose uptake into normal bovine eryihracyfes Normal bovine erythrocytes were incubated with serum from B. &v&infected calves. Under these conditions, a slight increase (less than two-fold) in glucose uptake into the normal bovine erythrocytes was observed (Table I). However this could be attributed to the rise in temperature of the experiment, as the erythrocytes exposed to infective serum were incubated at 37°C for 1 h and subsequently assayed at 22°C. Khetics ~~g~~cose entry into purasitized eryt~ir~cytes The kinetics of glucose influx into normal bovine erythrocytes could not be measured as the permeability of the cells to glucose was extremely low. Glucose influx in B. hovis-infected cells (53% parasitemia), using varying substrate concentrations, did not appear to saturate even at a concentration of glucose of 240 InM (Fig. 2), suggesting that glucose entered the parasitized bovine erythrocytes via simple diffusion.
, 60
120
180
240'
300
[glucose] (mM). FIG. 2. Effect of substrate concentration on rate of glucose influx into 8. hovis-infected bovine erythrocytes. The rate of [‘HIglucose influx (over 4 min) in a preparation of 53% B. bovis-infected bovine erythrocytes was assayed at 22’C as described in the Methods. with glucose concentrations ranging from IS to 240 mM. The values shown are the average of four replicate experiments.
loo-
80-
60-
40-
20-
Log [inhibitor] ($l) FIG. 3. Effect of inhibitors on glucose influx in B. bovis-infected bovine (53% parasitemia) and normal human erythrocytes. The effect of increasing concentrations (0.01-100 hi) of cytochalasin B and phloretin on uptake ofglucose (60 mM) in B. bovisinfected bovine erythrocytes (Fig. 3a 0, 0) and in normal human erythrocytes (5 mM) (Fig. 3b n , 0) over 4 min (B. 6oviF) or 1 min (human) intervals was determined as described in the Methods. Ceils were simultaneously exposed to both the inhibitor and the radiola~~led permeant prior to centrifugation through the oi1 layer. The values represent averages of triplicate ex~riments. Glucose uptake in the absence of any competing substrate was set at 100% activity. All other values were adjusted to this baseline.
13
GlucoseinfluxinB. bovis
Inhibition of glucose transport in parasitized erythrocytes To further characterize the properties of glucose
influx into 3. bovis-infected bovine erythrocytes, the effects of cytochalasin B, phloretin and phloridzin were investigated on a preparation of 53% parasitemia. Cytochalasin B was found to be without effect on the glucose influx into B. bovis-infected erythrocytes at concentrations of 0.1-100 PM (Fig. 3a). At the same range of concentrations, phloretin was found to inhibit glucose influx into B. bovis-infected erythrocytes by approximately 30% but was not shown to be concentration dependent. However, this ~on~ntration range of cytochalasin 3 and phloretin was sufficient to significantly inhibit glucose transport in normal human erythrocytes under the same experimentaf conditions (Fig. 3b). Conversely, phloridzin, over a concentration range of 10-500 ,UM, was demonstrated to significantly inhibit glucose uptake into B. bovisinfected bovine erythrocytes, whereas these concentrations did not affect glucose uptake into normal human cells (Fig. 4). relationship of glucose and nucleoside parasitized erythrocytes
tramport
in
The possible relationship of nucleoside and glucose transport in B. bovis-infected bovine erythrocytes was investigated using adenosine as an inhibitor of glucose uptake. Influx of glucose (60 mM) in B. bovis-infected bovine erythrocytes was found to be inhibited by
100
80
60
40
T
TABLET-PERCENT HUMAN AND B.
lNHIBITIONOFGLUCOSEINFLUXINTONORMAL bOYiS-INFECTED BOWNE ERYTHROCYTES BY ADENOSINE*
Normal human erythrocytes Adenosine % Inhibition (mM) 2 10 17
B. ~~v~-inf~~d bovine erythrocytes Adenosine (mM)
% Inhibition
2.5 10 15
62 -f: 6 57 f 6 57 f 3
0 0 7f2
* The influx of glucose into normal human erythrocytes was measured using 5 mM glucose. For B. bo~j~-infected bovine erythrocytes, the concentration of glucose was 60 mM. The values represented are the mean i S.D.of three replicates.
adenosine at concentrations between 2.5 and 15 mM (Table 2). A similar concentration range of adenosine (2-17 mM) had no effect on glucose uptake into normal human erythrocytes although the concentration of glucose used in the assay (5 IIIM)was significantly less than that used for B. bovis-infected cells (Table 2). Estimation
of cellular volumes
The cellular volumes of parasitized and uninfected bovine erythrocytes in the pellets beneath the oil in the assays were calculated from cell counts and intracellular volumes (Table 3). The presence of the Babesia parasite was not found to induce any change in the volume of the bovine erythrocyte, in contrast to the maIaria1 parasite in human erythrocytes which increased the cell volume (Gero, A. M. & Upston, J. M., unpublished data). The enhancement in B. bovisinfected erythrocyte membrane permeability, therefore, did not appear to be due to an alteration of host cell volume. TABLE
~-CELLULAR
VOLUMES OF PARASITIZEDAND NORMAL BOVINE~R~T~R~~T~S
Volume (fl)
Cell type
Normal bovine erythrocytes B. bovis-infected bovine erythrocytes?
20
37.9 f 2.5 (7)* 37.2 f 2.3 (3)
* The values in parentheses refer to the number of determinations used to calculate the average cellular volume l S.D (in A, lo-” X iitre). t Values represented correspond to parasitemias of 90%.
E -2
-3
0
1
2
3
DISCUSSION Log [Phloridzin} (PM). FIG. 4. Effect of phloridzin on glucose uptake in B. bovisinfected bovine and normal human erythrocytes. The effect
of phloridzin (0.01-I mM) on glucose (60 mm) uptake (over 4 min) in B. bovis-infected bovine erythrocytes (A) and normal human erythrocytes (a) was assayed as described for Fig. 3. Again, the values represent three replicate experiments.
Bovine erythrocytes, which are virtually impermeable to glucose, were shown to display dramatic increases in glucose permeability following infection with B. bovis. The results of kinetic analyses appeared to provide evidence for a non-saturable mode of glucose ‘transport’ as there was no departure from linearity even at concentrations of glucose 70-fold higher than
74
J. M. UPSTONand A. M. GERO
normal bovine plasma levels (3.5 RIM) (Fig. 2). However, not all criteria required for a simple diffusion model were satisfied. Simple diffusion is defined by the following: it is dependent on the maintenance of a concentration gradient, net movement is from a region of high concentration to a lower one, there is a low temperature dependence, the process is not saturable and there is no competition for substrate by structurally related molecules (She~an, 1988). Glucose uptake into parasitized bovine cells was found to be non-saturable and was not affected by increasing concentrations of established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin, at the concentrations shown to be effective in human erythrocytes (Fig. 3). Although phloretin reduced glucose influx into B. b&s-infected cells to approximately 70% of the control, the inhibition was not concentration-dependent over the concentration range tested. These data suggest that glucose permeability in B. bovis-infected erythrocytes is different, with respect to cytochalasin B and phloretin inhibition, from the normal human red cell glucose transport. Glucose influx, in B. bovis-infected cells was, however, significantly inhibited by phloridzin and adenosine, suggesting deviation from the simple diffusion model. In comparison, the transport of the amino acid, Lgiutamine, into the normal human erythrocyte has also been reported to be saturable, whilst t-glutamine influx into Plasmodium fulciparum-infected human erythrocytes was found to be a non-saturable process (Elford, Haynes, Chulay & Wilson, 1985; Ellory, Elford & Newbold, 1988). In addition the nonsaturable transport of L-glutamine was demonstrated to be specifically inhibited by piperine, artemetin and artesunate, whereas the uninfected erythrocyte transport remained unaffected. These results appear to be characteristically similar to those obtained for glucose influx into B. b&s-infected erythrocytes. suggesting that the mechanisms whereby Plasmodium and Babesia modify the membrane of their host cells to allow nutrient permeability may be analogous. Further, our data suggest that the component of glucose uptake found in B. b&s-infected bovine erythrocytes behaves differently to that found in normal mammalian cells. Phloridzin, at concentrations greater than 10 PM, effectively inhibited glucose influx into the parasitized cells, whilst the same concentration range of phloridzin did not inhibit glucose influx into normal human erythrocytes (Fig. 4). Phloridzin has been demonstrated to have antibabesial activity in in vitro cultures of B. bovis, with a IDSo (50% inhibitory dose) of 50 puw (Nott, Gero, O’Sullivan & Bagnara, unpublished data). Kutner, Breuer, Ginsburg & Cabantchik (1987) have also demonstrated that phloridzin effectively inhibits intraerythrocytic malarial growth in in vitro cultures of P. falciparum and have suggested that the antimalarial action may be associated in part with inhibition of solute transfer through membrane alterations induced
in the host cell membrane by the intracellular parasite, or via phloridzin acting directly with a parasite constituent. Our data with phloridzin again suggest that both intraerythrocytic parasites induce host cell membrane alterations to allow increased permeability. It could be envisaged that permeability changes in the membrane of parasite-infected erythrocytes proceeded through utilization of specific transport sites which were present, but not functionally active in the uninfected cell, such as reutilization of foetal calf red cell transporters. However, no glucose transport into newborn calf erythrocytes has been demonstrated (Kim, 1983) diminishing the possibility of the parasite activating foetal transport sites in the host cell to increase permeability. In addition, uninfected bovine cells did not increase in their permeability to glucose upon exposure to serum from infected calves, thereby suggesting that serum components were not responsible for the increased permeability of the B. bovisinfected cells to glucose. Adenosine was found to inhibit glucose influx in B. bovis-infected bovine erythrocytes suggesting an interaction of adenosine with the new or altered component of glucose ‘transport’ in these cells (Table 2). Similarly in B. rodhaini-infected rat erythrocytes, adenosine was found to competitively inhibit glucose uptake (Barry, 1982). In normal human erythrocytes both the glucose and nucleoside transport proteins migrate as a complex on sodium dodecyl sulphate polyacrylamide gel electrophoresis (band 4.5) and were found to have an apparent molecular weight of 4.5,00@-65,000. Despite the fact that the two proteins have not yet been separated, they could be discriminated on the basis of selective inhibitor binding (Paterson & Cass, 1985; Wheeler & Hinkle, 1985) and recognition by monoclonal antibodies (Tai & CarterSu, 1988). A recent study of the two proteins has revealed that nucleosides can interact with the glucose transporter of human erythrocytes (Jarvis, 1988). However, the glucose and nucleoside transporters are not simultaneously active in all mammalian species. For example, adult pig erythrocytes are impermeable to sugars, but retain functional nucleoside transport (Young, Paterson & Henderson, 1985). In comparison, there is no nucleoside transport into the bovine erythrocyte and, as demonstrated above, although the bovine erythrocyte glucose transport protein is functional, it transports this sugar at a rate of approximately l/2000 that of human erythrocytes (Hoos, Tarpley & Regen, 1972). The dramatic increase in glucose influx, which we have demonstrated in B. bovis-infected bovine erythrocytes, was found to be accompanied by induction of nucleoside transport (Gero 1989) and thus the possible reIationship of glucose and nucleoside transport in Lf. bovis-infected bovine cells was explored. The data in Table 2 indicate a possible relationship between adenosine and glucose transport, as adenosine (2.5-l 5 mM) appeared to inhibit glucose influx in B. bovis-infected cells. Three concentrations
Glucose
15
influx in B. bovis
of adenosine within a small range were chosen but an ID,, such as in Fig. 3 was not attempted. Further work on this phenomenon will be investigated but is not within the scope of this paper. Although adenosine was found to inhibit glucose transport in B. bovisinfected bovine erythrocytes (Table 2), the converse did not occur, as glucose (at concentrations up to 120 mM) was demonstrated to be without effect on adenosine transport (1 PM) (Gero, A. M., unpublished). Our results suggest, however, that adenosine interacts with the new or altered component of glucose ‘transport’ in B. bovis-infected bovine erythrocytes. Invasion of bovine erythrocytes with B. bovis did not appear to induce any change in the volume of the infected cell (Table 3). In comparison, the presence of mature intraerythrocytic forms of P. falciparum (trophozoites and schizonts) has been shown to increase the volume of infected human cells (Gero, A. M. & Upston, J. M., unpublished data). It is not known whether an increase in the volume of parasitized erythrocytes, due to changes in the membrane structure, could, therefore, account for alterations in membrane permeability in Plasmodium infections. However, the absence of an alteration in the bovine erythrocyte volume upon infection with B. bovis suggested that other mechanisms of increasing membrane permeability may operate in Babesiu infections. An alternative suggestion, to explain the mechanism whereby glucose (and other nutrients) permeability is altered by invasion of the bovine erythrocyte by B. bovis, may be misadjustment between phospholipid and protein in the erythrocyte membrane which has been used to account for the altered membrane properties of Plasmodium-infected erythrocytes (Schwartz, Olson, Raventos-Suarez, Yee, Heath, Lubin & Nagel, 1987). Increased permeability of parasitized cells could arise if parasite encoded proteins, embedded in the host cell membrane, did not form tight seals with the hydrocarbon chain of the phospholipids in the membrane (Ginsburg & Stein, 1987). Permeability changes apparent in Bubesia infections are similarly thought to arise through alterations in the phospholipid organization of the host cell membrane (Wright, Goodger & Clark, 1988) as B. bovis-infected bovine erythrocyte membranes have been found to contain parasite proteins (James, Montenegro-James, Fajfar-Whetstone, Montealegre, Erickson & Ristic, 1987). Additionally, B. bovisinfected bovine erythrocytes selectively bind to Heparin-Sepharose columns (Goodger, Mahoney & Wright, 1983), again suggesting that the membranes of the infected cells contain altered components. In conclusion, we have provided evidence that dramatic increases in glucose permeability occur in bovine erythrocytes upon infection with B. bovis. These data together with previous studies on rodent babesias (Rickard, 1969, Homewood et al., 1975; Momen, 1979; Barry, 1982), suggest that the babesia
parasite is able to alter the membrane permeability of the host cell to increase glucose uptake for its metabolic needs. The ‘transport’ mechanism whereby glucose enters B. bovis-infected erythrocytes appears to be characteristically different from that normally found in uninfected bovine and human erythrocytes. Also, adenosine was found to inhibit glucose influx in B. bovis-infected cells suggesting an interaction of the new or altered component of glucose ‘transport’ and the induced nucleoside transport. Further work in this area would elucidate whether the new or altered ‘transport’ of glucose is a leak, pore or an altered transporter in the parasitized erythrocyte. In addition, characterization of glucose (and other nutrients) influx mechanisms in infected cells and viable parasites, freed from the host cell membrane, may help to determine modes whereby parasite derived proteins are inserted into the membrane of the infected cell and whether changes in permeability occur due to alterations in the membrane structure. Acknowledgements-We wish to thank Dr I. G. Wright, Division of Tropical and Animal Production, CSIRO, Qld, for provision of Babesia bovis-infected erythrocytes and serum from infected calves and W. J. O’Sullivan for his interest. This work was supported by a grant to AMG from the National Health and Medical Research Council of Australia and the UNDP/World Bank/WHO Special Programme for the Research and Training in Tropical Diseases.
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