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Differences in glucose transport between bloodstream and procyclic forms of Trypanosoma brucei rhodesiense
Molecular and Biochemical Parasitology, 47 ( 1991) 73-82 © 1991 Elsevier Science Publishers B.V. /0166-6851/91/$03.50 ADONIS 016668519100077F
73
MOLBIO 01543
Differences in glucose transport between bloodstream and procyclic forms of Trypanosoma brucei rhodesiense Teresita M u n o z - A n t o n i a 1., Frank F. Richards I and Elisabetta Ullu ~'2 Yale MacArthur Centerfor Molecular Parasitology, JDepartment of lnternal Medicine and 2Cell Biology, Yale University School of Medicine, New Haven, CT, U.S.A. (Received 20 September 1990; accepted 10 January 1991)
In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitorN-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ions and is inhibited by phlorizin, an inhibitor of Na+-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na÷-independent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23/tM) than the bloodstream carrier (apparent Km 237 tiM). Key words: Trypanosome; Glucose transport; Inhibitor
Introduction
African trypanosomes of the Trypanosoma brucei subgroup are transmitted from mammal to mammal by their insect vector, the tsetse fly. During the trypanosome life cycle there are dramatic changes in morphology and metabolism [1]. Within their mammalian host, bloodstream trypanosomes are completely dependent on o-glucose for their energy supply, since they are not able to oxidise fatty acids or amino acids. When the tsetse fly ingests infected mammalian blood, the trypanosomes migrate into the insect midgut where they differentiate into procyclic forms. In this enCorrespondence address: Teresita Munoz-Antonia, Yale MacArthur Center for Molecular Parasitology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St New Haven CT 06515, U.S.A. Abbreviations: BS, bloodstream trypanosomes; PC, procyclic trypanosomes; 2-DOG, 2-deoxyglucose; NEM, N-ethylmaleimide.
vironment glucose is scarce, and amino acids such as proline constitute the main energy source [2]. As part of their adaptation to their new environment, the procyclic forms express a tricarboxylic acid cycle and a fully active mitochondrion appears. The trypanosomes ultimately migrate to the fly's salivary gland to complete their life cycle. Studies on glucose uptake in African trypanosomes have so far been restricted to the bloodstream forms. Available evidence suggests that a carrier molecule mediates o-glucose transport across the plasma membrane [3-5]. The transport of D-glucose is the rate limiting step in D-glucose metabolism [3]. Indeed, hexokinase and phosphofructokinase do not appear to play a regulatory role in glycolysis in T. brucei [6,7]. As outlined above, bloodstream and procyclic trypanosomes markedly differ in their requirement for glucose as energy source. In order to examine the possibility that glucose transport is regulated during differentiation from bloodstream to procyclic stage, we assessed whether cultured pro-
74
cyclic cells of T. b. rhodesiense were capable of transporting glucose against a concentration gradient. Our results suggest that these cells are indeed equipped with a specific glucose carrier. Kinetic measurements and inhibitor studies point to the possibility that different carrier molecules mediate D-glucose transport in bloodstream and procyclic cells. Materials and Methods
Cells. T.b. rhodesiense YTAT 1.1 were used in these studies. The procyclic forms were grown at 28°C in Cunningham's medium [8] supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS; Gibco, Grand Island, NY), 2 mM glutamine (Gibco) and 1× antibiotic-antimycotic (Gibco). For transport assays we used Cunningham's medium without any supplements and without sugars or proline (assay medium). Unless otherwise indicated, this assay medium was the medium used in all of our experiments. Cells grown to mid-log phase (57 × 106) were centrifuged for 5 min at 2500 × g in a clinical centrifuge and subsequently washed in assay medium. Bloodstream forms were grown in SpragueDawley rats. The blood was collected by cardiac puncture and the trypanosomes purified by passage through a DEAE-52 column as described [9], except that assay medium was used throughout the isolation procedure. Bloodstream trypanosomes were kept at 37°C in assay medium supplemented with 1% FCS prior to initiation of the transport experiments. Chemicals. The following compounds were obtained from Sigma Chemical Co., (St. Louis, MO): D-mannose, 2-deoxyglucose, D-glucosamine, Dglucose, N-acetyl-D-glucosamine, fructose, galactose, L-glucose, 3-o-methylglucose, N-ethylmaleimide (NEM) and phlorizin. Compounds not soluble in assay medium were resuspended in ethanol. The amount of ethanol in the assay medium never exceeded 1%. Transport assay. Trypanosomes were resuspended at 2 × 108 cells m1-1 in assay medium. To start the assay, 100/11 aliquots of the cell suspension were added to Eppendorf tubes containing droplets
with 70 pmol of 2-deoxy-D-[1,2-3H]glucose (2DOG; 30.2 Ci mmol-1; New England Nuclear, Wilmington, DE) dissolved in 100/11 of the assay medium, floating in a 700/tl cushion of a 2:1 mixture of dibutylphthalate:dioctylphthalate (d=l.030 g ml-1)(Aldrich Chemical Co., Milwaukee, WI). Incubations were done at 28°C for procyclic trypanosomes and at 37°C for bloodstream trypanosomes. After incubation, the Eppendorf tubes were centrifuged at 15 000 rev./min for 30 s in a microfuge to separate the trypanosomes (density = 1.067 g ml l) [10] from their aqueous environment (density = 1.01 g ml-~). After centrifugation, the assay medium (with the free 3H-labelled sugars) and the dibutylphthalate:dioctylphthalate cushion were aspirated, and the bottom of the Eppendorf tube containing the cell pellet was excised with a scalpel. The cell pellet was dissolved with 1% SDS, Optifluor (Packard Instrument Co., Downers Grove, IL) was added, and the samples were counted in a Beta Counter (Pharmacia LKB, Gaithersburg, MD). Background uptake was determined by measuring the uptake of 70 pmol of [ 1-3H(N)]-L-glucose (20.0 Ci mmol-l;NEN) under the same conditions. All transport determinations were done in duplicate. To test for inhibitors of 2-DOG transport, cells were preincubated for 10 min at the appropriate temperature with various compounds. After preincubation, transport assays were performed as described above. As a control for non-specific uptake or cell damage, uptake of [all]L-glucose under the same conditions was tested. [3H]2-deoxyglucose uptake in the presence of inhibitors was calculated by substracting the uptake of [3H]L-glucose under the same conditions. This value was divided by the uptake of [3H]2-deoxyglucose without inhibitors and multiplied by 100, to obtain the percentage of [3H]2-deoxyglucose uptake in the presence of a given inhibitor. To measure whether 2-DOG transport was Na +ion-dependent, a Na+-free buffer [11] was used instead of assay medium (which contains approximately 80 mM Na +ions) for uptake determinations. This buffer consisted of 100 mM choline chloride/2 mM KC1/1 mM MgCI2/1 mM CaCI2/10 mM Hepes, pH 7.5. The Na +containing buffer consisted of 100 mM NaC1/2 mM KCI/1 mM M g C l j l mM CaClJl 0 mM Hepes, pH 7.5. [3H]2-Deoxyglucose uptake in
75
the presence of different NaC1 concentrations was calculated by substracting the uptake of [3H]L-glucose under the same conditions. This value was divided by the uptake of [3H]2-deoxyglucose in assay medium and multiplied by a 100, to obtain the percentage of [3H]2-deoxyglucose uptake in the presence of different NaC1 concentrations. Hexokinase activity. The amount ofphosphorylated 2-[3H]DOG was determined in the following manner. Following 2-[3H]DOG uptake under standard conditions, cellswere centrifuged through 100 /11 of acetic acid (d=1.045 g ml 1) placed at the bottom of the dibutylphthalate:dioctylphthalate cushion. The assay medium and the dibutylphthalate:dioctylphthalate cushion were aspirated, and the acetic acid was left to evaporate. The cell pellets were then extracted with 5% TCA, TCA was removed with ether and the free and phosphorylated forms of the sugars were separated by thin layer chromatography (TLC) using a solvent system of a 66/31/1.5 mixture of isobutyric acid:H20:NH4OH. [ 12]. The sugars were detected on the TLC plates by fluorography using Autofluor (National Diagnostic, Manville, NJ). Scrapings from the TLC plates were counted in a Beta Counter (Pharmacia LKB) and used to determine the amounts of phosphorylated and unphosphorylated 2- [3H] DOG. lntracellular space determinations. The volume of the intracellular space of bloodstream and procyclic trypanosomes was determined according to Zilberstein and Dwyer [ 13]. Briefly, trypanosomes were resuspended at a concentration of 1.4 × 109 cells m1-1. From this cell suspension 0.4 ml was added to 1.2 ml of assay medium containing either 3H20 (15 ltCi ml-l)(Amersham Corp., Arlington Heights, IL.) and [3H]L-glucose (1.4/zCi ml -~) or 3H20 ( 1 5 / t C i ml -I) alone. After 3 min at the appropriate temperature, the cell suspensions were centrifuged for 1 min at 15 000 rev./min. The supernatant was aspirated and the cell pellet dissolved with 1% SDS and counted as described for transport experiments. Results
The ability of procyclic trypanosomes to transport glucose was examined using [3H]2-deoxyglu-
cose (2-DOG), a glucose analog that can be phosphorylated, but not further metabolised. This compound has been previously shown to be a suitable kinetic probe to assay glucose transport in various trypanosomatids, since no significant difference between uptake of D-glucose and 2-DOG is observed [3, 13]. As a mean to separate extracellular from intracellular sugar (which consists of free sugar plus the 2-DOG phosphorylated at the 6 position) we used rapid centrifugation through a medium of defined density as described in Materials and Methods. The time course of 2-DOG uptake by procyclic trypanosomes is shown in Fig. 1.2-DOG was taken up rapidly and reached plateau levels within 2 min, as shown previously for bloodstream trypanosomes [4]. In contrast, L-glucose did not accumulate within the procyclic cells at anytime point, thus demonstrating that 2-DOG transport was stereospecific. These initial experiments pointed to the possibility that procyclic trypanosomes are indeed equipped with a glucose carrier. Next the transport mechanism for 2-DOG were compared for procyclic and bloodstream trypanosomes. Towards this end we tested the sensitivity of D-glucose uptake in both cell types to 2 known inhibitors of glucose carriers, NEM and phlorizin. For these experiments, procyclic and bloodstream trypanosomes were preincubated for 10 min with
120
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Fig. 1. Time course of [3H]2-DOG uptake by procyclic trypanosomes. Uptake of [3H]2-DOG and [3H]L-glucose was determined as described in Materials and Methods. Each points represents the average of 2 determinations. ([2) [3H]2DOG; (0) [3H]L-glucose.
76
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the inhibitors before the transport assay was initiated. Uptake of L-glucose was measured under the same conditions. Cell motility was checked throughout the duration of the experiment. As shown in Fig. 2, we found that NEM completely inhibited 2-DOG uptake both in procyclic and bloodstream trypanosomes. Since 1 mM NEM covalently modifies free sulphydryl groups, these data suggests that a protein molecule could participate in glucose transport in trypanosomes. Phlorizin, an inhibitor of Na+-dependent glucose transport in mammalian cells considerably inhibited 2DOG transport in bloodstream trypanosomes (Fig. 2), confirming an earlier report by Gruenberg et al. [3]. In contrast, no effect on 2-DOG transport in procyclic trypanosomes was observed (Fig. 2). This latter result was confirmed with phlorizin con-
centrations up to 5 mM (data not shown). Higher concentrations could not be tested because they were toxic to the cells as determined by loss of motility and non-specific uptake of L-glucose. These data suggest the existence of different mechanisms of glucose transport in bloodstream and procyclic trypanosomes. To further confirm the Na + dependency of the bloodstream glucose carrier, 2-DOG uptake was measured in the presence and absence of Na + ions. This was accomplished by substituting choline ions for sodium ions in an assay buffer previously used to determine the Na +requirements of other glucose transport systems [11]. Because bloodstream trypanosomes lost their motility after 20 min incubation in the choline buffer system, we could not fully deplete the cells ofNa + ions by extensive pre-
77
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80
70
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~ 40
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60
80
100
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Fig. 3. Effect of Na÷ions on [3H]2-DOGtransport by trypanosomes.During the transport assay, assay medium was substituted for a choline chloride buffer (0 mM NaCI) containing different amounts of NaC1. Uptake was measured for 30 s as described in Materials and Methods. Each point represents the average of 2 determinations. Percentage 2-[3H]DOGuptake is given by: (Uptake of 2-DOG in choline buffer) - (Uptake of L-glucose in choline buffer) x 100 (Uptake of 2-DOG in Assay Media) - (Uptake of L-glucose in Assay Media)
incubations. Therefore, cell pellets were resuspended in the appropriate buffer without preincubations and allowed to uptake 2-DOG for 30 s. Using this procedure, uptake of 2-DOG by procyclic trypanosomes was not affected by the absence of Na + ions (Fig. 3). In contrast we observed an inhibition of 2-DOG transport in bloodstream trypanosomes which was proportional to the concentration of Na + ions in the assay medium (Fig. 3). These results confirm that glucose uptake in bloodstream trypanosomes is Na÷-dependent. We also examined the specificity o f procyclic and bloodstream glucose carriers by assaying the ability o f other sugars to prevent transport of 2DOG. Competition experiments were performed by adding 0.5 m M non-radioactive sugars at the start o f the 2-DOG transport assay. D-glucose, D-
glucosamine, N-acetyl-D-glucosamine and D-mannose inhibited 2-DOG uptake by more than 75% both in procyclic and bloodstream trypanosomes (data not shown). This is an indication that these sugars utilise the same transporter. In contrast, galactose, fructose, and 3-o-methylglucose did not compete with 2-DOG transport in either procyclic or bloodstream trypanosomes suggesting that they were not transported by the same carrier. To further characterise glucose carriers of procyclic and bloodstream trypanosomes we compared the kinetics of glucose uptake. However, since 2-DOG is a substrate for hexokinase, we first needed to determine if hexokinase activity was rate limiting before meaningful kinetic comparisons could be made. Furthermore, we needed a method to rapidly inactivate hexokinase in order to accu-
78
rately evaluate phosphorylation achieved during the transport assay. To this end, cells were allowed to transport 2-DOG for 15, 30 and 60 s and rapidly centrifuged to the bottom of tubes containing 100 /zl of acetic acid overlayed with the defined density medium used throughout the experiments. The extent of phosphorylation of 2-DOG was then analyzed by thin layer chromatography. The results of these determinations showed that phosphorylation of 2-DOG did not appear to be rate limiting neither in bloodstream nor in procyclic trypanosomes (Table I). Having established that hexokinase activity showed no significant differences between the 2 trypanosome stages we were able to directly analyze the kinetics of 2-DOG transport. It has been shown that there is no difference in the kinetics of uptake between 2-DOG and D-glucose in bloodstream trypanosomes [3]. For this reason, and because 2-DOG is toxic to bloodstream trypanosomes at high concentrations we decided to use 2[3H]DOG as a tracer for uptake using different Dglucose concentrations to dilute the labelled compound. Fig. 4 shows the rate of glucose uptake as a function of glucose concentrations from 0 to 100 mM. The uptake curve continued to rise up to 500 mM glucose (data not shown), suggesting that at very high ligand concentrations additional transport phenomena are observed. From double reciprocal plots apparent Km and Vmaxwere calculated for 3 independent experiments. In all experiments, the affinity for glucose was an order of magnitude TABLE I 2-DOG phosphorylation
Bloodstreams
Procyclics
Time (s)
2-DOG uptake (pmol)
%2-DOG6-P
15 30 60
1.0 2.4 5.7
95 86 78
15 30 60
0.8 2.0 4.6
83 75 79
2-DOG phosphorylation in bloodstream and procyclic trypanosomes. 2-DOG uptake was determined as described in Materilals and Metheods. Duplicate samples were analyzed using thin layer chromatography and the TLC scrapings used to determine the amount of phosphorylated 2-DOG (2-DOG6-P) relative to 2-DOG at different time intervals.
higher in procyclic trypanosomes (Km 23-+ 2/.tM) than in bloodstream trypanosomes (Kin 237 + 18 /.tM). Conversely, bloodstream trypanosomes had a higher capacity to transport glucose (Vmax 110 nmol min-l(mg protein) -1 than procyclic trypanosomes (Vma x 11.4 nmol min-l(mg protein) -1. Based on these determinations it appears that the affinity for glucose and its maximum rate of transport are different in the 2 trypanosome stages. It must be stressed that the calculated Km and Vmaxrepresent the contribution of many factors and, therefore, are only indicative values. Next we determined the intracellular concentration of 2-DOG (which represents the free 2-DOG and the 2-DOG-6-P). In order to do this we measured the intracellular volume of bloodstream and procyclic trypanosomes by calculating the difference in volume of freely distributed 3H20 and the excluded sugar [3H]L-glucose. The L-glucose excluded from the cells under these conditions has been found to correspond to the extracellular volume determined by [3H]inulin [3]. Using this method we determined that bloodstream trypanosomes have an intracellular volume of 2.0/11 (108 cells) ~, and procyclic trypanosomes of 1.6 #1 (108 cells) -~. These values are comparable to those obtained by other investigators for bloodstream trypanosomes [5,14]. Based on the assumption that intracellular glucose can be distributed throughout all intracellular water space, the intracellular concentration of 2-DOG was then calculated. The results of these determinations are shown in Table II. For both procyclic and bloodstream trypanosomes, the intracellular concentration of 2-DOG was higher than that of the extracellular milieu (0.35 /1tool). Discussion In initial experiments on glucose transport using T. b. rhodesiense cells we found that the assay con-
ditions are of primary importance to obtain meaningful and reproducible results. Indeed, it has been reported that when non-physiological conditions are used to isolate bloodstream trypanosomes, biochemical changes occur in the parasite [15]. We screened a number of buffer systems, including Krebs-Ringer and Na-phosphate, for their ability to maintain the motility of bloodstream and procyclic
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Fig. 4. Concentration dependence of [3H]2-DOG uptake in bloodstream and procyclic trypanosomes. Uptake of [3H]2-DOG was measured after 30 s in the presence of different D-glucose concentrations. Each point represents the average of 2 determinations. (A) Procyclic trypanosomes; (B) Bloodstream trypanosomes. Inset: double reciprocal plots of uptake kinetics. TABLE II Intracellular volume of trypanosomes
Bloodstreams Procyclics
Cell volume
Intracellular 2-DOG
2.0 ill(108cells)-' 1.6/ll(108cells)-'
20.0//rnol 15.6/zmol
Intracellular volume was calculated for bloodstream and procyclic trypanosomes using [3H] L-glucose and 3H20 as markers for extracellular and intracellular volume respectively. The intracellular 2-DOG concentration, calculated after 3 min of uptake, was 0.35/tmol.
trypanosomes unaltered upon 30 min incubation at the appropriate temperature. None of the above buffers was found adequate; however cell motility was
unaffected by incubating bloodstream and procyclic trypanosomes in Cunningham's medium [8] lacking sugars but supplemented with 1% foetal calf serum. As a method to separate free from intracellular sugar for transport studies, we decided to employ a rapid centrifugation technique by which trypanosomes are sedimented through a medium of defined density which allows the cells, but not the incubation fluid, to be recovered at the bottom of the tubes. Attempts to separate the cells by filtration as described by Zilberstein and Dwyer [13] failed, probably because T. b. rhodesiense cells are damaged by the procedure. Using the assay conditions described above, we demonstrate that culture procyclic trypanosomes
80 uptake 2-DOG against a concentration gradient. D-glucose, but not L-glucose, inhibited uptake of 2DOG, indicating that the glucose transporter is stereo specific for the i~ form of glucose and that, as reported in earlier studies [3], D-glucose and 2-DOG are transported by the same carrier. Inhibition of 2-DOG uptake by incubation of procyclic cells with NEM suggests a protein moiety in glucose uptake. When the properties of the glucose transporter systems of bloodstream and procyclic trypanosomes were compared, we found that they share some similarities in substrate specificity. However, our data also suggest that different glucose carrier molecules function in bloodstream and procyclic trypanosomes. The most compelling evidence is the differential inhibition by phlorizin, which suggests that only bloodstream glucose transport is Na+-dependent. Consistent with this observation is the requirement of Na + ions for glucose transport in bloodstream but not in procyclic trypanosomes. In trypanosomes a specialised organelle, the glycosome, contains the enzymes of the glycolytic pathway, including hexokinase [16]. The glycosome can be distinguished morphologically and functionally, and its membrane represents a true permeability barrier [16]. It is important to point out that the process we have measured is transport across the plasma membrane and across the glycosomal membrane. Evidence for this is the rapid and high level of phosphorylation of 2-DOG, an event which takes place mostly in the glycosome. Unfortunately, intact glycosomes are difficult to isolate, therefore it is not possible to experimentally dissociate transport across these 2 membranes. We established that the relative levels of hexokinase activity are similar in bloodstream and procyclic trypanosomes (Table I). Since hexokinase activity is not a rate limiting step in glucose uptake, differences between procyclic and bloodstream trypanosomes in glucose uptake kinetics must be intrinsic either to the specific glucose carrier molecules on the cell surface or perhaps to other yet to be defined glucose carrier, which might translocate glucose from the cytoplasm into the glycosome. Since the rate of phosphorylation by hexokinase is rapid enough to prevent accumulation of nonphosphorylated 2-DOG, the rate of uptake can be taken as a measure of unidirectional transport. The
apparent Km measured in procyclic trypanosomes was 23/.tM. This is comparable to what has been described in another trypanosomatidae, the Leishmania donovani promastigotes, whose glucose transporter has a Km of 24 tiM [ 13]. It can be speculated that both of these organisms, which live in environments where glucose is scarce, need a high affinity transporter to allow them to transport whatever glucose is available. Bloodstream trypanosomes live in a glucose rich environment, therefore they would not need a transporter of such high affinity. The affinity of their transporter (apparent K,, 237/tM), is still high enough to ensure their survival in the mammalian blood where the sugar concentration is 5 mM. The Km reported by us for bloodstream trypanosomes is lower than the previously reported Kms for bloodstream trypanosomes (1 mM [3]; 3-4 mM [4,5]). One possible explanation for this discrepancy is that the Km was calculated as a function of glucose concentrations between 0 and 100/tM. In addition, since some of the assays repoeted by others were done at 20 ° C [5] whereas we used physiological temperatures, it is possible that the bloodstream trypanosome glucose transporter is affected by changes in temperature as has been reported in other systems [17]. On the other hand, the apparent Km of 237 ¢tM for the bloodstream trypanosome glucose transporter is consistent with the affinities reported for other Na+-driven glucose transporters [18]. The Na+-dependent glucose transporter uses the gradient of Na + ions between the extracellular and intracellular milieu as a free energy source for co-transport. Bloodstream trypanosomes posses in their plasma membrane a Na+/K + ATPase [19-21], which could be linked to this process. Under mammalian physiological conditions (140 mmol Na+l-l), Na + co-transporters are probably saturated, thus, transport can occur as soon as the sugar is bound. Although our data provides evidence for Na+-de pendent glucose uptake in bloodstream trypanosomes, we cannot exclude the possibility that 2 uptake systems are operating in these cells since inhibition of glucose transport by phlorizin in bloodstream trypanosomes was never 100%. The first system, unique to the bloodstream trypanosomes, appears to be Na+-dependent. This Na+-de pendent system would be readily available, and en-
81 sure the b l o o d s t r e a m t r y p a n o s o m e s its vital glucose supply. A different s y s t e m , possibly c o m m o n to b l o o d s t r e a m and procyclic t r y p a n o s o m e s , is N a += independent. This system has a higher affinity for glucose guaranteeing glucose uptake in environments where glucose is scarce. Other cell types (for e x a m p l e h u m a n skeletal muscle and kidney), have been s h o w n to have m o r e than one kind o f glucose transport m e c h a n i s m [22]. It can be speculated that different t r y p a n o s o m e ' s life stages could have different glucose uptake m e c h a n i s m s to c o m p e n s a t e for the different e n v i r o n m e n t s they live in. It is not surprising that the bloodstream t r y p a n o s o m e s m i g h t have 2 kinds o f glucose transporter to ensure their supply o f glucose in the different environments they encounter. After this paper was submitted an independent report on glucose uptake in procyclic t r y p a n o s o m e s was published [23]. Our results and those by Nielsen and Parsons are in agreement.
8 9 10 11
12
13 14
Acknowledgements 15 W e are grateful to Drs. Christian Tschudi, Peter Mason, B u d d y U l l m a n and Jeffrey R a d d i n g for critical reading o f the manuscript. W e wish to thank L o u i s e C a m e r a - B e n s o n for her technical assistance. This w o r k was supported in part by N I H grants A I 0 7 8 6 0 and A I 08614 and the John D. and Catherine T. M a c A r t h u r Foundation.
References 1 Vickerman, K. (1965) Polymorphism and mitochondrial activity in sleeping sickness trypanosomes. Nature 208, 762-766. 2 Mordue, W., Goldsworthy, G.J., Brady, J., and Blaney, W.M. (1980) Energy metabolism. In: Insect Physiology. (Mordue, W.,Goldsworthy, G.J., Brady, J., and Blaney, W.M., eds.), pp 1-12. Blackwell, Oxford. 3 Gruenberg, J., Sharma, P.R., and Deshusses, J. (1978) oglucose transport in Trypanosoma brucei: Eur. J. Biochem. 89, 461-469. 4 Game, S., Holman, G., and Eisenthal, R. (1978) Sugar transport in Trypanosoma brucei: a suitable kinetic probe. FEBS Lett. 194, 126-130. 5 Eisenthal, R., Game, S., and Holman, G.D. (1989) Specificity and kinetics of hexose transport in Trypanosoma brucei. Biochim. Biophys. Acta 985, 81-89. 6 Seed, J.R., and Baquero, M.A. (1965) The characterization of hexokinase from Trypanosoma rhodesiense and Trypanosoma gambiense. J. Protozool. 12,427-432. 7 Nwagwu, N., and Opperdoes, F.R. (1982). Regulation of
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18 19
20
glycolysis in Trypanosoma brucei: hexokinase and phosphofructokinase activity. Acta Trop. 39, 61-72. Cunningham, I. (1977) New culture media for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325-329. Lanham, S.M., and Godfrey, D.G. (1970) Isolation of salivarian trypanosomes from man and other animals using DEAE-cellulose. Exp. Parasitol. 28,521-524. Grab, D.J., and Bwayo, J.J. (1982) Isopycnic isolation of African trypanosomes on percoll gradients formed in situ. Acta Trop. 39,363-366. Hediger, M.A., Ikeda, T., Coady, M., Gundersen,C.B., and Wright, E.M. (1987) Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes. Proc. Natl. Acad. Sci., USA 84, 2634-2637. Tsoboi, K.K.,andPetriciani, J.C. (1975) Concentrative accumulation (active transport) of 2-deoxy-D-glucose in primate fibroblasts. Biochem. Biophys. Res. Commun. 62, 587--593. Zilberstein, D., and Dwyer, D.M. (1984) Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 12, 327-336. Voorheis, H.P., and Martin B.R. (1980) Swell dyalisis demonstrates that adenylate cyclase in Trypanosoma brucei is regulated by calcium ions. Eur. J. Biochem. 113, 223-227. Londsdale, J.D., and Grab, D.J. (1987) Purification of African trypanosomes can cause biochemical changes in the parasite. J. l:hotozool. 34, 405-408. Visser, N., Opperdoes, F.R., and Borst, P. ( 1981) Subcellular compartmentation of glycolytic intermediates in Trypanosoma brucei. Eur. J. Biochem. 18,521-526, Bro!-Laroche, E., Serrano, M.A., Delhomme, B., and Alvarado, F. (1986) Temperature sensitivity and substrate specificity of two distinct Na+-activated o-glucose transport systems in guinea pig jejunal brush border membrane vesicles. J. Biol. Chem. 261,6168~5176. Malo, C. (1988) Kinetic evidence for heterogeneity in Na÷o-glucose cotransport systems in the normal human foetal small intestine. Biochim. Biophys. Acta 938, 18 l-188. Rovis, L., and Baekkeskov, S. (1980). Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes. Parasitology 80, 507524. Voorheis, H.P., Gale, J.S., Owen, M.J., and Edwards, W. (1979) The isolation and characterization of the plasma membrane from Trypanosoma brucei. Biochem. J.180,
11-24. 21 Mancini, P.E., Strickler,J.E., and Patton, C.L. 1982. Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei. Biochim. Biophys. Acta 688,399-4 l 0. 22 Permutt, M.A., Koranyi, L., Keller, K., Lacy, P.E., Scharp, D.W., and Mueckler, M. (1989) Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA. Proc. Natl. Acad. Sci. USA 86, 8688-8692. 23 Parsons, M. and Nielsen B. (1990) Active transport of 2-deoxy-o-glucose in Trypanosoma brucei procyclic forms. Mol. Biochem. Parasitol. 42, t97-204.
73
MOLBIO 01543
Differences in glucose transport between bloodstream and procyclic forms of Trypanosoma brucei rhodesiense Teresita M u n o z - A n t o n i a 1., Frank F. Richards I and Elisabetta Ullu ~'2 Yale MacArthur Centerfor Molecular Parasitology, JDepartment of lnternal Medicine and 2Cell Biology, Yale University School of Medicine, New Haven, CT, U.S.A. (Received 20 September 1990; accepted 10 January 1991)
In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitorN-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ions and is inhibited by phlorizin, an inhibitor of Na+-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na÷-independent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23/tM) than the bloodstream carrier (apparent Km 237 tiM). Key words: Trypanosome; Glucose transport; Inhibitor
Introduction
African trypanosomes of the Trypanosoma brucei subgroup are transmitted from mammal to mammal by their insect vector, the tsetse fly. During the trypanosome life cycle there are dramatic changes in morphology and metabolism [1]. Within their mammalian host, bloodstream trypanosomes are completely dependent on o-glucose for their energy supply, since they are not able to oxidise fatty acids or amino acids. When the tsetse fly ingests infected mammalian blood, the trypanosomes migrate into the insect midgut where they differentiate into procyclic forms. In this enCorrespondence address: Teresita Munoz-Antonia, Yale MacArthur Center for Molecular Parasitology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St New Haven CT 06515, U.S.A. Abbreviations: BS, bloodstream trypanosomes; PC, procyclic trypanosomes; 2-DOG, 2-deoxyglucose; NEM, N-ethylmaleimide.
vironment glucose is scarce, and amino acids such as proline constitute the main energy source [2]. As part of their adaptation to their new environment, the procyclic forms express a tricarboxylic acid cycle and a fully active mitochondrion appears. The trypanosomes ultimately migrate to the fly's salivary gland to complete their life cycle. Studies on glucose uptake in African trypanosomes have so far been restricted to the bloodstream forms. Available evidence suggests that a carrier molecule mediates o-glucose transport across the plasma membrane [3-5]. The transport of D-glucose is the rate limiting step in D-glucose metabolism [3]. Indeed, hexokinase and phosphofructokinase do not appear to play a regulatory role in glycolysis in T. brucei [6,7]. As outlined above, bloodstream and procyclic trypanosomes markedly differ in their requirement for glucose as energy source. In order to examine the possibility that glucose transport is regulated during differentiation from bloodstream to procyclic stage, we assessed whether cultured pro-
74
cyclic cells of T. b. rhodesiense were capable of transporting glucose against a concentration gradient. Our results suggest that these cells are indeed equipped with a specific glucose carrier. Kinetic measurements and inhibitor studies point to the possibility that different carrier molecules mediate D-glucose transport in bloodstream and procyclic cells. Materials and Methods
Cells. T.b. rhodesiense YTAT 1.1 were used in these studies. The procyclic forms were grown at 28°C in Cunningham's medium [8] supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS; Gibco, Grand Island, NY), 2 mM glutamine (Gibco) and 1× antibiotic-antimycotic (Gibco). For transport assays we used Cunningham's medium without any supplements and without sugars or proline (assay medium). Unless otherwise indicated, this assay medium was the medium used in all of our experiments. Cells grown to mid-log phase (57 × 106) were centrifuged for 5 min at 2500 × g in a clinical centrifuge and subsequently washed in assay medium. Bloodstream forms were grown in SpragueDawley rats. The blood was collected by cardiac puncture and the trypanosomes purified by passage through a DEAE-52 column as described [9], except that assay medium was used throughout the isolation procedure. Bloodstream trypanosomes were kept at 37°C in assay medium supplemented with 1% FCS prior to initiation of the transport experiments. Chemicals. The following compounds were obtained from Sigma Chemical Co., (St. Louis, MO): D-mannose, 2-deoxyglucose, D-glucosamine, Dglucose, N-acetyl-D-glucosamine, fructose, galactose, L-glucose, 3-o-methylglucose, N-ethylmaleimide (NEM) and phlorizin. Compounds not soluble in assay medium were resuspended in ethanol. The amount of ethanol in the assay medium never exceeded 1%. Transport assay. Trypanosomes were resuspended at 2 × 108 cells m1-1 in assay medium. To start the assay, 100/11 aliquots of the cell suspension were added to Eppendorf tubes containing droplets
with 70 pmol of 2-deoxy-D-[1,2-3H]glucose (2DOG; 30.2 Ci mmol-1; New England Nuclear, Wilmington, DE) dissolved in 100/11 of the assay medium, floating in a 700/tl cushion of a 2:1 mixture of dibutylphthalate:dioctylphthalate (d=l.030 g ml-1)(Aldrich Chemical Co., Milwaukee, WI). Incubations were done at 28°C for procyclic trypanosomes and at 37°C for bloodstream trypanosomes. After incubation, the Eppendorf tubes were centrifuged at 15 000 rev./min for 30 s in a microfuge to separate the trypanosomes (density = 1.067 g ml l) [10] from their aqueous environment (density = 1.01 g ml-~). After centrifugation, the assay medium (with the free 3H-labelled sugars) and the dibutylphthalate:dioctylphthalate cushion were aspirated, and the bottom of the Eppendorf tube containing the cell pellet was excised with a scalpel. The cell pellet was dissolved with 1% SDS, Optifluor (Packard Instrument Co., Downers Grove, IL) was added, and the samples were counted in a Beta Counter (Pharmacia LKB, Gaithersburg, MD). Background uptake was determined by measuring the uptake of 70 pmol of [ 1-3H(N)]-L-glucose (20.0 Ci mmol-l;NEN) under the same conditions. All transport determinations were done in duplicate. To test for inhibitors of 2-DOG transport, cells were preincubated for 10 min at the appropriate temperature with various compounds. After preincubation, transport assays were performed as described above. As a control for non-specific uptake or cell damage, uptake of [all]L-glucose under the same conditions was tested. [3H]2-deoxyglucose uptake in the presence of inhibitors was calculated by substracting the uptake of [3H]L-glucose under the same conditions. This value was divided by the uptake of [3H]2-deoxyglucose without inhibitors and multiplied by 100, to obtain the percentage of [3H]2-deoxyglucose uptake in the presence of a given inhibitor. To measure whether 2-DOG transport was Na +ion-dependent, a Na+-free buffer [11] was used instead of assay medium (which contains approximately 80 mM Na +ions) for uptake determinations. This buffer consisted of 100 mM choline chloride/2 mM KC1/1 mM MgCI2/1 mM CaCI2/10 mM Hepes, pH 7.5. The Na +containing buffer consisted of 100 mM NaC1/2 mM KCI/1 mM M g C l j l mM CaClJl 0 mM Hepes, pH 7.5. [3H]2-Deoxyglucose uptake in
75
the presence of different NaC1 concentrations was calculated by substracting the uptake of [3H]L-glucose under the same conditions. This value was divided by the uptake of [3H]2-deoxyglucose in assay medium and multiplied by a 100, to obtain the percentage of [3H]2-deoxyglucose uptake in the presence of different NaC1 concentrations. Hexokinase activity. The amount ofphosphorylated 2-[3H]DOG was determined in the following manner. Following 2-[3H]DOG uptake under standard conditions, cellswere centrifuged through 100 /11 of acetic acid (d=1.045 g ml 1) placed at the bottom of the dibutylphthalate:dioctylphthalate cushion. The assay medium and the dibutylphthalate:dioctylphthalate cushion were aspirated, and the acetic acid was left to evaporate. The cell pellets were then extracted with 5% TCA, TCA was removed with ether and the free and phosphorylated forms of the sugars were separated by thin layer chromatography (TLC) using a solvent system of a 66/31/1.5 mixture of isobutyric acid:H20:NH4OH. [ 12]. The sugars were detected on the TLC plates by fluorography using Autofluor (National Diagnostic, Manville, NJ). Scrapings from the TLC plates were counted in a Beta Counter (Pharmacia LKB) and used to determine the amounts of phosphorylated and unphosphorylated 2- [3H] DOG. lntracellular space determinations. The volume of the intracellular space of bloodstream and procyclic trypanosomes was determined according to Zilberstein and Dwyer [ 13]. Briefly, trypanosomes were resuspended at a concentration of 1.4 × 109 cells m1-1. From this cell suspension 0.4 ml was added to 1.2 ml of assay medium containing either 3H20 (15 ltCi ml-l)(Amersham Corp., Arlington Heights, IL.) and [3H]L-glucose (1.4/zCi ml -~) or 3H20 ( 1 5 / t C i ml -I) alone. After 3 min at the appropriate temperature, the cell suspensions were centrifuged for 1 min at 15 000 rev./min. The supernatant was aspirated and the cell pellet dissolved with 1% SDS and counted as described for transport experiments. Results
The ability of procyclic trypanosomes to transport glucose was examined using [3H]2-deoxyglu-
cose (2-DOG), a glucose analog that can be phosphorylated, but not further metabolised. This compound has been previously shown to be a suitable kinetic probe to assay glucose transport in various trypanosomatids, since no significant difference between uptake of D-glucose and 2-DOG is observed [3, 13]. As a mean to separate extracellular from intracellular sugar (which consists of free sugar plus the 2-DOG phosphorylated at the 6 position) we used rapid centrifugation through a medium of defined density as described in Materials and Methods. The time course of 2-DOG uptake by procyclic trypanosomes is shown in Fig. 1.2-DOG was taken up rapidly and reached plateau levels within 2 min, as shown previously for bloodstream trypanosomes [4]. In contrast, L-glucose did not accumulate within the procyclic cells at anytime point, thus demonstrating that 2-DOG transport was stereospecific. These initial experiments pointed to the possibility that procyclic trypanosomes are indeed equipped with a glucose carrier. Next the transport mechanism for 2-DOG were compared for procyclic and bloodstream trypanosomes. Towards this end we tested the sensitivity of D-glucose uptake in both cell types to 2 known inhibitors of glucose carriers, NEM and phlorizin. For these experiments, procyclic and bloodstream trypanosomes were preincubated for 10 min with
120
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100
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~
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&
2-DCG L-gluc0se
20
.
4
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Fig. 1. Time course of [3H]2-DOG uptake by procyclic trypanosomes. Uptake of [3H]2-DOG and [3H]L-glucose was determined as described in Materials and Methods. Each points represents the average of 2 determinations. ([2) [3H]2DOG; (0) [3H]L-glucose.
76
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0
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I
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Fig. 2. Effect of different inhibitors on [JH]2-DOG uptake by bloodstream and procyclic trypanosomes. Cells were pre-incubated for 10 min in the presence of the inhibitors prior to the addition of radiolabeled sugars. Uptake was measured for 1 min after the addition of the radiolabeled sugars as described in Materials and Methods. Percentage 2-[3H]DOG uptake is given by: (Uptake of 2-DOG + inhibitor) - (Uptake of L-glucose + inhibitor) x 100 (Uptake of 2-DOG) - (Uptake of L-glucose)
the inhibitors before the transport assay was initiated. Uptake of L-glucose was measured under the same conditions. Cell motility was checked throughout the duration of the experiment. As shown in Fig. 2, we found that NEM completely inhibited 2-DOG uptake both in procyclic and bloodstream trypanosomes. Since 1 mM NEM covalently modifies free sulphydryl groups, these data suggests that a protein molecule could participate in glucose transport in trypanosomes. Phlorizin, an inhibitor of Na+-dependent glucose transport in mammalian cells considerably inhibited 2DOG transport in bloodstream trypanosomes (Fig. 2), confirming an earlier report by Gruenberg et al. [3]. In contrast, no effect on 2-DOG transport in procyclic trypanosomes was observed (Fig. 2). This latter result was confirmed with phlorizin con-
centrations up to 5 mM (data not shown). Higher concentrations could not be tested because they were toxic to the cells as determined by loss of motility and non-specific uptake of L-glucose. These data suggest the existence of different mechanisms of glucose transport in bloodstream and procyclic trypanosomes. To further confirm the Na + dependency of the bloodstream glucose carrier, 2-DOG uptake was measured in the presence and absence of Na + ions. This was accomplished by substituting choline ions for sodium ions in an assay buffer previously used to determine the Na +requirements of other glucose transport systems [11]. Because bloodstream trypanosomes lost their motility after 20 min incubation in the choline buffer system, we could not fully deplete the cells ofNa + ions by extensive pre-
77
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90 L~a V I,a_
I
~
80
70
6o Bloodstreams 50 Pr'ocyc lic5
¢
4 0
0 20
~ 40
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60
80
100
(mPI)
Fig. 3. Effect of Na÷ions on [3H]2-DOGtransport by trypanosomes.During the transport assay, assay medium was substituted for a choline chloride buffer (0 mM NaCI) containing different amounts of NaC1. Uptake was measured for 30 s as described in Materials and Methods. Each point represents the average of 2 determinations. Percentage 2-[3H]DOGuptake is given by: (Uptake of 2-DOG in choline buffer) - (Uptake of L-glucose in choline buffer) x 100 (Uptake of 2-DOG in Assay Media) - (Uptake of L-glucose in Assay Media)
incubations. Therefore, cell pellets were resuspended in the appropriate buffer without preincubations and allowed to uptake 2-DOG for 30 s. Using this procedure, uptake of 2-DOG by procyclic trypanosomes was not affected by the absence of Na + ions (Fig. 3). In contrast we observed an inhibition of 2-DOG transport in bloodstream trypanosomes which was proportional to the concentration of Na + ions in the assay medium (Fig. 3). These results confirm that glucose uptake in bloodstream trypanosomes is Na÷-dependent. We also examined the specificity o f procyclic and bloodstream glucose carriers by assaying the ability o f other sugars to prevent transport of 2DOG. Competition experiments were performed by adding 0.5 m M non-radioactive sugars at the start o f the 2-DOG transport assay. D-glucose, D-
glucosamine, N-acetyl-D-glucosamine and D-mannose inhibited 2-DOG uptake by more than 75% both in procyclic and bloodstream trypanosomes (data not shown). This is an indication that these sugars utilise the same transporter. In contrast, galactose, fructose, and 3-o-methylglucose did not compete with 2-DOG transport in either procyclic or bloodstream trypanosomes suggesting that they were not transported by the same carrier. To further characterise glucose carriers of procyclic and bloodstream trypanosomes we compared the kinetics of glucose uptake. However, since 2-DOG is a substrate for hexokinase, we first needed to determine if hexokinase activity was rate limiting before meaningful kinetic comparisons could be made. Furthermore, we needed a method to rapidly inactivate hexokinase in order to accu-
78
rately evaluate phosphorylation achieved during the transport assay. To this end, cells were allowed to transport 2-DOG for 15, 30 and 60 s and rapidly centrifuged to the bottom of tubes containing 100 /zl of acetic acid overlayed with the defined density medium used throughout the experiments. The extent of phosphorylation of 2-DOG was then analyzed by thin layer chromatography. The results of these determinations showed that phosphorylation of 2-DOG did not appear to be rate limiting neither in bloodstream nor in procyclic trypanosomes (Table I). Having established that hexokinase activity showed no significant differences between the 2 trypanosome stages we were able to directly analyze the kinetics of 2-DOG transport. It has been shown that there is no difference in the kinetics of uptake between 2-DOG and D-glucose in bloodstream trypanosomes [3]. For this reason, and because 2-DOG is toxic to bloodstream trypanosomes at high concentrations we decided to use 2[3H]DOG as a tracer for uptake using different Dglucose concentrations to dilute the labelled compound. Fig. 4 shows the rate of glucose uptake as a function of glucose concentrations from 0 to 100 mM. The uptake curve continued to rise up to 500 mM glucose (data not shown), suggesting that at very high ligand concentrations additional transport phenomena are observed. From double reciprocal plots apparent Km and Vmaxwere calculated for 3 independent experiments. In all experiments, the affinity for glucose was an order of magnitude TABLE I 2-DOG phosphorylation
Bloodstreams
Procyclics
Time (s)
2-DOG uptake (pmol)
%2-DOG6-P
15 30 60
1.0 2.4 5.7
95 86 78
15 30 60
0.8 2.0 4.6
83 75 79
2-DOG phosphorylation in bloodstream and procyclic trypanosomes. 2-DOG uptake was determined as described in Materilals and Metheods. Duplicate samples were analyzed using thin layer chromatography and the TLC scrapings used to determine the amount of phosphorylated 2-DOG (2-DOG6-P) relative to 2-DOG at different time intervals.
higher in procyclic trypanosomes (Km 23-+ 2/.tM) than in bloodstream trypanosomes (Kin 237 + 18 /.tM). Conversely, bloodstream trypanosomes had a higher capacity to transport glucose (Vmax 110 nmol min-l(mg protein) -1 than procyclic trypanosomes (Vma x 11.4 nmol min-l(mg protein) -1. Based on these determinations it appears that the affinity for glucose and its maximum rate of transport are different in the 2 trypanosome stages. It must be stressed that the calculated Km and Vmaxrepresent the contribution of many factors and, therefore, are only indicative values. Next we determined the intracellular concentration of 2-DOG (which represents the free 2-DOG and the 2-DOG-6-P). In order to do this we measured the intracellular volume of bloodstream and procyclic trypanosomes by calculating the difference in volume of freely distributed 3H20 and the excluded sugar [3H]L-glucose. The L-glucose excluded from the cells under these conditions has been found to correspond to the extracellular volume determined by [3H]inulin [3]. Using this method we determined that bloodstream trypanosomes have an intracellular volume of 2.0/11 (108 cells) ~, and procyclic trypanosomes of 1.6 #1 (108 cells) -~. These values are comparable to those obtained by other investigators for bloodstream trypanosomes [5,14]. Based on the assumption that intracellular glucose can be distributed throughout all intracellular water space, the intracellular concentration of 2-DOG was then calculated. The results of these determinations are shown in Table II. For both procyclic and bloodstream trypanosomes, the intracellular concentration of 2-DOG was higher than that of the extracellular milieu (0.35 /1tool). Discussion In initial experiments on glucose transport using T. b. rhodesiense cells we found that the assay con-
ditions are of primary importance to obtain meaningful and reproducible results. Indeed, it has been reported that when non-physiological conditions are used to isolate bloodstream trypanosomes, biochemical changes occur in the parasite [15]. We screened a number of buffer systems, including Krebs-Ringer and Na-phosphate, for their ability to maintain the motility of bloodstream and procyclic
79
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,
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Fig. 4. Concentration dependence of [3H]2-DOG uptake in bloodstream and procyclic trypanosomes. Uptake of [3H]2-DOG was measured after 30 s in the presence of different D-glucose concentrations. Each point represents the average of 2 determinations. (A) Procyclic trypanosomes; (B) Bloodstream trypanosomes. Inset: double reciprocal plots of uptake kinetics. TABLE II Intracellular volume of trypanosomes
Bloodstreams Procyclics
Cell volume
Intracellular 2-DOG
2.0 ill(108cells)-' 1.6/ll(108cells)-'
20.0//rnol 15.6/zmol
Intracellular volume was calculated for bloodstream and procyclic trypanosomes using [3H] L-glucose and 3H20 as markers for extracellular and intracellular volume respectively. The intracellular 2-DOG concentration, calculated after 3 min of uptake, was 0.35/tmol.
trypanosomes unaltered upon 30 min incubation at the appropriate temperature. None of the above buffers was found adequate; however cell motility was
unaffected by incubating bloodstream and procyclic trypanosomes in Cunningham's medium [8] lacking sugars but supplemented with 1% foetal calf serum. As a method to separate free from intracellular sugar for transport studies, we decided to employ a rapid centrifugation technique by which trypanosomes are sedimented through a medium of defined density which allows the cells, but not the incubation fluid, to be recovered at the bottom of the tubes. Attempts to separate the cells by filtration as described by Zilberstein and Dwyer [13] failed, probably because T. b. rhodesiense cells are damaged by the procedure. Using the assay conditions described above, we demonstrate that culture procyclic trypanosomes
80 uptake 2-DOG against a concentration gradient. D-glucose, but not L-glucose, inhibited uptake of 2DOG, indicating that the glucose transporter is stereo specific for the i~ form of glucose and that, as reported in earlier studies [3], D-glucose and 2-DOG are transported by the same carrier. Inhibition of 2-DOG uptake by incubation of procyclic cells with NEM suggests a protein moiety in glucose uptake. When the properties of the glucose transporter systems of bloodstream and procyclic trypanosomes were compared, we found that they share some similarities in substrate specificity. However, our data also suggest that different glucose carrier molecules function in bloodstream and procyclic trypanosomes. The most compelling evidence is the differential inhibition by phlorizin, which suggests that only bloodstream glucose transport is Na+-dependent. Consistent with this observation is the requirement of Na + ions for glucose transport in bloodstream but not in procyclic trypanosomes. In trypanosomes a specialised organelle, the glycosome, contains the enzymes of the glycolytic pathway, including hexokinase [16]. The glycosome can be distinguished morphologically and functionally, and its membrane represents a true permeability barrier [16]. It is important to point out that the process we have measured is transport across the plasma membrane and across the glycosomal membrane. Evidence for this is the rapid and high level of phosphorylation of 2-DOG, an event which takes place mostly in the glycosome. Unfortunately, intact glycosomes are difficult to isolate, therefore it is not possible to experimentally dissociate transport across these 2 membranes. We established that the relative levels of hexokinase activity are similar in bloodstream and procyclic trypanosomes (Table I). Since hexokinase activity is not a rate limiting step in glucose uptake, differences between procyclic and bloodstream trypanosomes in glucose uptake kinetics must be intrinsic either to the specific glucose carrier molecules on the cell surface or perhaps to other yet to be defined glucose carrier, which might translocate glucose from the cytoplasm into the glycosome. Since the rate of phosphorylation by hexokinase is rapid enough to prevent accumulation of nonphosphorylated 2-DOG, the rate of uptake can be taken as a measure of unidirectional transport. The
apparent Km measured in procyclic trypanosomes was 23/.tM. This is comparable to what has been described in another trypanosomatidae, the Leishmania donovani promastigotes, whose glucose transporter has a Km of 24 tiM [ 13]. It can be speculated that both of these organisms, which live in environments where glucose is scarce, need a high affinity transporter to allow them to transport whatever glucose is available. Bloodstream trypanosomes live in a glucose rich environment, therefore they would not need a transporter of such high affinity. The affinity of their transporter (apparent K,, 237/tM), is still high enough to ensure their survival in the mammalian blood where the sugar concentration is 5 mM. The Km reported by us for bloodstream trypanosomes is lower than the previously reported Kms for bloodstream trypanosomes (1 mM [3]; 3-4 mM [4,5]). One possible explanation for this discrepancy is that the Km was calculated as a function of glucose concentrations between 0 and 100/tM. In addition, since some of the assays repoeted by others were done at 20 ° C [5] whereas we used physiological temperatures, it is possible that the bloodstream trypanosome glucose transporter is affected by changes in temperature as has been reported in other systems [17]. On the other hand, the apparent Km of 237 ¢tM for the bloodstream trypanosome glucose transporter is consistent with the affinities reported for other Na+-driven glucose transporters [18]. The Na+-dependent glucose transporter uses the gradient of Na + ions between the extracellular and intracellular milieu as a free energy source for co-transport. Bloodstream trypanosomes posses in their plasma membrane a Na+/K + ATPase [19-21], which could be linked to this process. Under mammalian physiological conditions (140 mmol Na+l-l), Na + co-transporters are probably saturated, thus, transport can occur as soon as the sugar is bound. Although our data provides evidence for Na+-de pendent glucose uptake in bloodstream trypanosomes, we cannot exclude the possibility that 2 uptake systems are operating in these cells since inhibition of glucose transport by phlorizin in bloodstream trypanosomes was never 100%. The first system, unique to the bloodstream trypanosomes, appears to be Na+-dependent. This Na+-de pendent system would be readily available, and en-
81 sure the b l o o d s t r e a m t r y p a n o s o m e s its vital glucose supply. A different s y s t e m , possibly c o m m o n to b l o o d s t r e a m and procyclic t r y p a n o s o m e s , is N a += independent. This system has a higher affinity for glucose guaranteeing glucose uptake in environments where glucose is scarce. Other cell types (for e x a m p l e h u m a n skeletal muscle and kidney), have been s h o w n to have m o r e than one kind o f glucose transport m e c h a n i s m [22]. It can be speculated that different t r y p a n o s o m e ' s life stages could have different glucose uptake m e c h a n i s m s to c o m p e n s a t e for the different e n v i r o n m e n t s they live in. It is not surprising that the bloodstream t r y p a n o s o m e s m i g h t have 2 kinds o f glucose transporter to ensure their supply o f glucose in the different environments they encounter. After this paper was submitted an independent report on glucose uptake in procyclic t r y p a n o s o m e s was published [23]. Our results and those by Nielsen and Parsons are in agreement.
8 9 10 11
12
13 14
Acknowledgements 15 W e are grateful to Drs. Christian Tschudi, Peter Mason, B u d d y U l l m a n and Jeffrey R a d d i n g for critical reading o f the manuscript. W e wish to thank L o u i s e C a m e r a - B e n s o n for her technical assistance. This w o r k was supported in part by N I H grants A I 0 7 8 6 0 and A I 08614 and the John D. and Catherine T. M a c A r t h u r Foundation.
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