Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

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MOLECULAR

ELSEVIER

iZHEMrcAL PARASITOLOGY Molecular

and Biochemical

Parasitology

70 (1995) 59-69

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter J. Conrad Schwab ay1,Mohammed Afifi Afifi a, Giuseppe Pizzorno b, Robert E. Handschumacher b, Keith A. Joiner a,* ADepartment of Internal Medicine, Infectious Diseases Section, Yale Unitiersity School of Medicine, 333 Cedar Street, New Hal’en. CT 06520-8022, USA h Department of Pharmacology, Yale Unirlersity School of Medicine, New Hatlen, CT 06.520-8022. USA Received 30 August 1994; accepted

16 December

1994

Abstract Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [3H]adenosine I.&fold within 30 s, approx. half of the [3H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [ 14C]Inosine, [ “C]hypoxanthine and [ 3H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC,, approx. 0.7 PM), but not nitrobenzylthioinosine (15 PM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 FM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [ ‘Hladenosine and [ 14C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. go&ii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purines may also be present. Kevvords:

Purine; Protozoan; Nucleoside transport: Dipyridamole;

Nitrobenzylthioinosine

1. Introduction Infection

with the protozoan

parasite Toxoplasma disease in the

gondii may progress to life-threatening

Abbre[aiations: AISS, Artificial Intracellular salt solution; HBSS, Hanks Balanced Salt Solution; NBTI, nitrobenzylthioinosine (6-[(4-nitrobenzyl)thio]-9-P-D_ribofuranosylpurine); TCA, trichloroacetic acid; TLC, thin-layer chromatography * Corresponding author. Tel.: (l-203) 785.4140; Fax: (l-203) 785-3864. ’ Present address: SUNY Health Science Center, 750 E. Adams Street, Syracuse, NY 13210, USA 0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166.6851(95100005-4

immunocompromised host [I]. Because current therapy is unsatisfactory, new therapeutic drug targets are being sought. One area of considerable therapeutic potential is the purine utilization pathway of this obligate intracellular organism [2]. Prior studies demonstrated that T. gondii, which is capable of synthesizing pyrimidine nucleosides de novo [3-51, is a purine auxotroph [6,7]. Therefore, purine needed for growth must be salvaged from the host cell [6]. Furthermore, T. gondii can convert adenine to guanine nucleotides, but not the reverse

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and Biochemical Parasitology 70 (1995) 59-69

[7]. Thus only adenine, adenosine, hypoxanthine, or inosine salvaged from the host cell can fulfill all of the purine requirements of T. go&ii. Of these, adenosine is the most efficiently salvaged by extracellular tachyzoites [7], probably due to an extremely high level of adenosine kinase activity [7,8]. Although the intracellular purine economy of T. go&i has been partially delineated [7], the crucial first step in utilization of adenosine or other purine substrates, permeation of the parasite plasma membrane, has not been previously examined. In mammalian cells, two principal types of nucleoside transporters have been characterized: non-concentrative facilitated diffusion transporters, and concentrative, sodium-dependent, active transporters [9,10] (reviewed in Ref. 11). Facilitated diffusion transporters are typically sensitive to nanomolar (or less frequently micromolar) concentrations of nitrobenzylthioinosine (NBTI), usually with comparable sensitivity to dipyridamole [ll]. Concentrative, active nucleoside transporters are characteristically resistant to NBTI and dipyridamole inhibition [lo]. Mammalian NBTI-sensitive cells express high affinity NBTIbinding sites that in general are felt to be equivalent to the nucleoside binding site of the carrier. The high-affinity binding site for dipyridamole, while overlapping with the NBTI and carrier nucleoside binding site, is not identical (reviewed in Ref. 111. Cells may simultaneously express several types of transporters [9,111. We have evaluated purine transport in T. gondii using labeled substrates and giving particular attention to the intracellular form of the transported substrate. We have identified a single dipyridamole-sensitive nitrobenzylthioinosine (IC,,, < I PM), (NBTI)-resistant (IC,,, approx. 15 PM) adenosine transporter in the plasma membrane of T. gondii tachzoites.

(Irvine, CA, USA); and [.5-3H]uridine from Moravek (Brea, CA, USA). Unlabeled nucleosides and other reagents were purchased from Sigma (St. Louis, MO, USA). Dipyridamole and NBTI were prepared as 20 mM and 15 mM stocks, respectively, in DMSO and stored in aliquots at -40°C. 2.2. Buffers and media VA-13 media (Irvine Scientific, Santa Ana, CA, USA) [7] with 3.5 mg ml-’ bovine serum albumin and 10 mM HEPES (pH 7.2) or Artificial Intracellular Salt Solution (AISS) buffer [12] with 20 mM HEPES (pH 7.2) were used in most experiments. VA-13 media reportedly extends the viability of extracellular tachyzoites [7], while AISS buffer is patterned after the ionic composition of the intracellular compartment 1121. Results from experiments using HBSS or Minimum Essential Media, alpha modification, were not significantly different from results with VA-13 or AISS, probably because incubations were brief and done at 24°C. Sodium-free (choline replacement) HBSS [13] was also used in some experiments. 2.3. Preparation

of parasites

Tachyzoites were harvested from the peritoneal fluid of mice infected for 3 or 4 days with the RH strain of T. gondii as described [14]. The adenosine kinase-deficient mutant of the RH strain, generated and characterized by Pfefferkorn and Pfefferkorn [8,15] was also used. Parasites were purified by passage through a 27 gauge needle [16] and 3 pm polycarbonate filter [ 171, washed twice by centrifugation at 1000 X g for 8 min, and resuspended at a cell density of approx. 3 X lo* tachyzoites ml-’ in incubation buffer. Parasites were not used for experiments if there was > 1% contamination with host cells, as assessed by visual inspection.

2. Materials and methods 2.1. Reagents

2.4. Measurement stop technique

of substrate

transport

by the oil-

[2-3H]Adenosine, [8-3H]adenine and [8-14C]inosine were from Amersham (Arlington Heights, IL, USA); [S-l4 Clhypoxanthine and [ 3~]~z~ were from NEN (Boston, MA, USA); [ r4C]inulin from ICN

Nucleoside or base uptake into parasites was measured by the oil-stop technique [18]. In most experiments, uptake was initiated by mixing 100 /Al of the parasite suspension in VA-13 or AISS buffer (results

J.C. Schwab et al. /Molecular

and Biochemical Parasitology 70 (1995) 59-69

were similar in the two buffer systems, and are presented interchangeably in the Results section) with an equal volume of labeled substrate in the same buffer. At subsequent intervals, triplicate 60-~1 aliquots of the cell suspension were applied to the top of 400-~1 microcentrifuge tubes containing 50 ~1 of 15% trichloroacetic acid (TCA) overlaid with 125 ~1 of a 84% 550 silicon fluid (Dow, Midland, MI, USA) 16% light paraffin oil (Fisher, Fair Lawn, NJ, USA) mixture. Parasites with incorporated radioactivity were then pelleted into the TCA layer by centrifugation in a Hill IMC microcentrifuge at 13 000 X g for 30 s. For replicate sampling at uptake intervals shorter than 30 s, oil-stop tubes were loaded, incubated, and centrifuged individually. The tubes were sliced through the oil layer and the quantity of radioactivity present in the top and bottom halves determined by liquid scintillation counting. The intracellular volume of the cells and the correction required for radioactivity present in extracellular media carried into the TCA layer by the pelleted cells were determined in parallel microcentrifuge tubes using [“H]H~o and [‘JC]inulin as described [18]. The mean parasite volume for the 10’ parasites typically used in each oil-stop tube was 0.111 k 0.006 ~1 cell water (n = 38). 2.5. Inhibition / competitire transport

blockade

of substrate

Inhibition of nucleoside transport was tested with NBTI and with dipyridamole. Parasites were either mixed with varying concentrations of these inhibitors for 15 min prior to addition of labeled substrate, or the parasites were mixed with labeled substrate in the presence of inhibitor. Adenosine efflux from parasites was also examined. For each timepoint, adenosine kinase-deficient tachyzoites were loaded with 300 ,uM [3H]adenosine for 60 s at room temperature in a total volume of 15 ~1. Efflux was initiated by adding 7.5 ~1 of this suspension to 150 ~1 of buffer with or without 10 PM dipyridamole on top of an oil-stop tube loaded in the microcentrifuge. Efflux was terminated by pelleting the parasites into the TCA layer. Parasiteassociated (TCA layer) radioactivity was then quantitated. In competitive inhibition experiments, 100 ~1 of

61

parasites were mixed with 20 /IM labeled substrate and varying concentrations (200-1600 PM final concentration) of unlabeled substrate. Transport/uptake was measured by the oil-stop technique. 2.6. Chromatography The form of radioactivity in the cell pellet fraction at different times was determined using thin-layer chromatography (TLC) or HPLC. TCA layers (50 ~1) containing disrupted parasites with incorporated label were extracted with an equal volume of 1 M trioctylamine in 1,1,2-trichlorotrifluoroethane (Freon). For TLC, samples were mixed with 5 mM unlabeled adenine, adenosine, hypoxanthine, inosine and ATP, and spotted in duplicate onto TLC plates (Merck silica gel 60, art. 5735), dried and developed with 58% isopropanol (v/v)/24% ethyl acetate/ 10% water/g% ammonium hydroxide [19]. Purines were localized under short-wavelength UV. Lanes were sectioned and radioactivity present in each region then quantitated by liquid scintillation counting. Results from TLC were confirmed with HPLC using a Cl8 reverse phase column (Whatman). The mobile phase consisted of 3% acetonitrile (v/v) in a 50 mM potassium phosphate buffer with a flow rate of 1 ml min-’ at 24°C. Absorbance of the effluent was monitored at 254 nm, and l-ml fractions were collected and counted for radioactivity. Observed retention times were: purine nucleotides, 3 min; hypoxanthine, 5 min; adenine, 8 min; inosine, 10 min; adenosine, 26 min. Purine nucleotides were analyzed using a linear gradient of sodium phosphate (pH 3.3) from 10 to 700 mM on a Partisil SAX10 column (25 cm X 4.6 mm) at 1.S ml min-’ at 24°C. Observed retention times were: AMP, 4 min; ADP, 30 min; ATP, 40 min; GMP, 19 min; GDP, 33 min; GTP, 46 min.

3. Results 3.1. Adenosine is rapidly transported into T. gondii tachyzoites and conrlerted to nucleotide Initial experiments focused on adenosine uptake since adenosine is the preferred substrate for purine

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and Biochemical Parasitology 70 (1995) 59-69

salvage by T. gondii [7]. Adenosine uptake by T. gondii was rapid. For extracellular [ 3H]adenosine concentrations ranging from 3 to 40 PM, the intracellular concentration of radioactivity exceeded the extracellular within 30 s (Fig. 1A). After 9 min, [ 3Hladenosine was concentrated about 20-fold within the tachyzoites. Since total uptake of radioactivity was measured, this apparent concentration of [ ‘Hladenosine within tachyzoites could reflect trapping of the adenosine by rapid metabolism, rather than active transport against a concentration gradient. TLC and HPLC analysis revealed that adenosine was rapidly converted to nucleotide form. In a representative experiment using 20 PM adenosine (Fig. lB), nucleotides represented the majority of intracellular label after 1 min and about 90% after 9 min. Analysis of the intracellular label by strong anionexchange HPLC (not shown) revealed the nucleotide fraction to be principally ATP, while guanine nucleotides were not appreciably labeled over this time interval. This result is consistent with the known extremely high level of adenosine kinase activity in this organism [7,8], and maintenance of a high ATP/ADP ratio in viable extracellular tachyzoites [7]. In contrast to the accumulation of nucleotides, the intracellular concentration of label present as free adenosine was equal to the extracellular [ 3H]adenosine concentration. This result suggests that adenosine is transported by facilitated diffusion rather than by a concentrative transporter. However, extremely rapid metabolism of adenosine after transport could obscure a concentrative transport process. Since adenosine uptake in sodium-free (choline replacement) HBSS was undiminished relative to HBSS buffer (not shown), sodium-dependent, concentrative transport does not appear to be a contributing mechanism. 3.2. Adenosine transport is non-concentrative in an adenosine kinase deficient mutant of T. gondii To characterize more completely the transport mechanism, it was necessary to minimize metabolism of the labeled adenosine by adenosine kinase. Uptake of adenosine was measured in an adenosine kinasedeficient mutant of T. gondii derived from the RH strain [&IS] in the presence of a higher extracellular adenosine concentration (200 PM). TLC analysis

1000

A 1 40 @A adsnosine

0

120

240

360

460

600

total _/(4B

Time (s)

1B

adenosine

0

120

240

360

460

600

Time (s)

Fig. 1. Uptake and metabolism of [3H]adenosine by RH strain T. gondii. (A) Uptake of [3H]adenosine by extracellular RH strain T. gondii tachyzoites from 3 ( n ), 10 CO), 20 ( A ) and 40 (0 ) PM initial extracellular adenosine concentration (spec. act. 1.7-0.13 Ci mmol-‘1. Parasites were mixed with adenosine, then at intervals separated from the media by centrifugation through oil and incorporated radioactivity assayed as described in Materials and methods. Data depicted are means + SD (less than symbol size for several datapoints) of triplicate determinations at each adenosine concentration and timepoint. (B) Form of incorporated label during uptake of adenosine. TLC was performed on cell pellets collected during uptake of 20 PM adenosine (extracellular concentration, - - - - -1, as described in Materials and methods. The total uptake of radioactivity (-. n -. -) reflected accumulation of 0) and free adenosine adenosine-derived nucleotide ( 0 A) in equilibrium with the media concentration. For (Aclarity, the low concentrations of adenine, inosine and hypoxanthine present in the cells are not shown.

revealed that 2.5% of the intracellular label was present as nucleotide at 0 s, 6.1% at 7 s and 9.4% at 15 s. The limited uptake and metabolism at the nominal O-s timepoint reflects the approx. 2-s inter-

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and Biochemical Parasitology

val before cells are separated from the aqueous media and enter the TCA in the oil-stop technique [20]. Intracellular adenosine rapidly equilibrated with but at no time exceeded the extracellular concentration, further supporting a non-concentrative facilitated diffusion transport system. 3.3.

Adenosine

high-capacity

is transported

by a low-affinity,

70 (1995) 59-69

(conditions which minimize metabolism of the label), the values were 230 PM and 25 pmol(p1 cell water per ’ (not shown). Based on the results presented in the previous section, the last figures best estimate the kinetics of this nucleoside transporter, since uptake reflects transport in the adenosine-kinase-deficient mutant studied under these conditions, but not the wild-type RH strain.

s>-

transport system

3.4. Transport of adenosine Initial adenosine uptake velocity was examined in both the adenosine kinase deficient-mutant and wild-type RH strain. These experiments, in which parasites were mixed with a fixed concentration of labeled adenosine and increasing concentrations of unlabeled adenosine, revealed that uptake was a saturable process (Fig. 2). Using values for the 15-s time point, the estimated K, and V,,, for adenosine uptake in the wild-type RH strain were 120 PM and 17 pmol (~1 cell water per s)-r and for the adenosine-kinase-deficient mutant were 150 PM and 21 pmol ( ~1 cell water per s1-l (Fig. 2, insets A,B). values for the adenosineWhen K, and V,,, kinase-deficient mutant were recalculated using substrate concentrations greater than 64 PM adenosine

Ol 0

63

200 Extracellular

400

600

adenosine (MM)

Fig. 2. Uptake of adenosine is saturable in the adenosine-kinasedeficient mutant. Uptake of adenosine in the adenosine-kinase-deficient mutant was determined at 15 s for concentrations ranging from 1-512 PM. Similar results were seen in experiments examining initial adenosine and inosine uptake by wild-type RH parasites. The insets show Lineweaver-Burk plots for (A) wild-type RH and (B) adenosine-kinase-deficient RH mutant.

into T. gondii is blocked

by dipyridamole

Inhibitors have been very useful in the characterization of transporter systems 1211. The specific and prototypic inhibitor of mammalian nucleoside transport is NBTI, which blocks most mammalian facilitated diffusion-type nucleoside transporters at nanomolar concentrations [9,111. In T. gondii tachyzoites, 15 PM NBTI (with and without 15 min pre-incubation) produced only a moderate reduction in adenosine uptake. As demonstrated in Fig. 3A, parasite uptake of 20 PM adenosine had already exceeded the concentration present in media in both control and NBTI conditions by 30 s. In contrast, 10 ,uM dipyridamole, a structurallyunrelated and less-specific inhibitor of nucleoside transport [ll], effectively blocked entrance of adenosine into the parasites (Fig. 3B). The extent of inhibition was similar for the wild-type RH strain and the mutant lacking adenosine kinase. Dipyridamole inhibition of adenosine transport was dose dependent, with an ICs,, of approx. 0.7 ,uM (Fig. 30, a value in the upper range of that reported for dipyridamole-sensitive nucleoside transporters in mammalian cells [ 111. Since the intracellular [ 3H]adenosine concentration (irrespective of form) did not approach the extracellular concentration even after 180 s (Fig. 3B), dipyridamole inhibition of T. gondii adenosine uptake presumably occurred at the level of transport rather than subsequent metabolism. In the later case, rapid equilibration of intracellular and extracellular adenosine radioactivity would have been expected. Additional evidence that dipyridamole blocked T. gondii membrane transport of adenosine was its inhibition of efflux of adenosine from dipyridamoletreated adenosine-kinase-deficient parasites (Fig. 3D). Although the combination of the adenosine kinase

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and Biochemical Parasitology 70 (1995) 59-69

deficient parasite and a high extracellular substrate concentration was chosen to minimize metabolism of the label, the asymmetric influx and efflux curves probably reflect metabolic trapping of adenosine. 3.5. Inosine, hypoxanthine and adenine transport across the T. gondii plasma membrane is minimally inhibited by dipyridamole To explore further the transport of potential purine substrates for T. gondii, we measured transport of

the inosine, hypoxanthine and adenine. Uptake of these substrates was rapid (Fig. 4). Within 15 s, intracellular levels of these substrates approached the extracellular level of 20 PM. After 30 s, 31% of inosine, 32% of hypoxanthine and 6% of adenine were converted to nucleotide form. Conversion of inosine to hypoxanthine (11% at 30 s) was also noted. However, as with adenosine, transport was non-concentrative. The apparent K, and V,,, for inosine transport at 15 s, conditions in which greater than 80% of the radioactivity was present as inosine,

1

240 z

0

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60

180

120

60 Time

Time (s)

160

(s)

C 4 ‘; 3 E ‘E %

100 60 60

9 z ; :

40

:

0

20

k

0

. . ..

..I

.

.l

.,....I

.

1

Dipyridamole (pM)

.

OY.,

....‘.l

10

-60

I., -30

0 Time

. 30

f 60

(s)

Fig. 3. Effect of inhibitors of adenosine transport in RH strain T. gondii tachyzoites. (Al Effect of 15 PM NBTI on T. gondii adenosine uptake (20 PM extracellular adenosine, - - - - - -l. Parasites were pre-treated with NBTI (0 ) or buffer (0) for at least 1.5 min prior to measuring adenosine uptake as described in Materials and methods. Data depict means k SD (less than symbol size for several datapoints) of a single experiment representative of 3 separate experiments. (B) Effect of 10 PM dipyridamole on T. gondii adenosine uptake. Extracellular parasites were pretreated with dipyridamole (A) or buffer (0) and adenosine uptake determined. Note that uptake in dipyridamole-treated parasites fails to reach the adenosine concentration of the extracellular media (20 PM, - - - - -), even at 180 s. Data depict means f SD (less than symbol size for several datapoints) of a single experiment representative of 3 separate experiments. (Cl Effect of increasing concentrations of dipyridamole on adenosine uptake. Uptake of adenosine at 30 s was determined in RH strain T. gondii in the presence of dipyridamole concentrations of O-10 PM. Data are means + SD of at least 3 independent experiments. No non-specific toxic effect of 10 /.LM dipyridamole on parasite viability was observed, as assessed by parasite morphology, Trypan blue dye exclusion, or ability to infect human foreskin fibroblast monolayers. (D) Dipyridamole blocks adenosine efflux from loaded cells. Adenosine kinase-deficient parasites were loaded with [3H]adenosine (300 FM extracellular concentration) for 60 s. To initiate efflux, 7.5-~1 aliquots were added to the top of a oil-stop tube containing 150 ~1 of buffer (1 : 21 final dilution) with (m W) or without (A -Aa) 10 /.LM dipyridamole. The parasites were then pelleted through the oil layer at different time intervals and total radioactivity in the pellet assayed as described in Materials and methods.

J.C. Schwab et al. / Molecular and Biochemical Parasitology 70 (1995) 59-69

were 81 PM and 5.5 pmol (~1 cell water per s>-‘, respectively (Fig. 4A, inset). In contrast to the results with adenosine, dipyridamole only inhibited transport of inosine, hypoxanthine and adenine to a limited extent (Fig. 4). Dipyridamole (10 PM) inhibited inosine, hypoxanthine and adenine uptake, measured at 30 s, by 21, 17 and 7%, respectively.

3.6. Competition but not adenosine

experiments suggest that inosine has two transport systems

B.

Hypoxanthine aol

We next focused on the substrate specificity of adenosine and inosine transport to determine if the above differences in dipyridamole inhibition reflected the presence of an additional transport system for purines other than adenosine. The purines inosine, hypoxanthine and adenine inhibited transport of adenosine (Fig. 5A), but uridine, thymidine and cytidine (not shown) did not. The substrates deoxyadenosine and formycin B which are poorly or non-metabolizable by mammalian cells [9] also competed with adenosine transport, minimizing the likelihood that inhibition occurred at the level of nucleoside or nucleotide phosphorylation or feedback inhibition. These results suggest that there is a common transport locus for purine nucleosides and bases. In contrast, neither the purines adenosine and adenine nor the pyrimidines uridine or thymidine substantially blocked inosine uptake (Fig. 5B). Only formycin B and hypoxanthine substantially inhibited transport of inosine. Subsequent experiments were conducted at short time intervals (3, 6 and 9 s) to minimize the likelihood that substrate metabolism influenced the substrate competition experiments. Inosine almost completely inhibited adenosine transport, whereas adenosine blocked inosine transport by a maximum of only 27%. To analyze the competition between inosine and adenosine for uptake, experiments were conducted in which uptake of either L3H]adenosine or [‘4C]inosine was measured in the presence of the alternative labeled substrate and in the presence or absence of dipyridamole (Fig. 50. Transport of [ 3H]adenosine was inhibited by [ “C]inosine, and was completely blocked by dipyridamole independent of the pres-

C.

Adenine 401

Fig. 4. Uptake of inosine. hypoxanthine and adenine by wild-type RH strain T. gondii and inhibition by dipyridamole. Uptake of 20 FM inosine (panel A), 20 PM hypoxanthine (panel B) and 20 PM adenine (panel C) by wild-type RH strain T. gondii was measured as described for adenosine. Fraction of total label for each timepoint and condition present as unmodified substrate represented by shaded areas (0); nucleotide by cross-hatched areas ( 0 ) and other metabolic product as clear areas (0 ). Data depict a single experiment representative of at least three independent experiments for each substrate. Inset in A shows a Lineweaver-Burk plot for inosine uptake analyzed at 15 s.

ence or absence of [‘4C]inosine. In contrast, transport of [14C]inosine was not substantially decreased by L3H]adenosine, and the effect of dipyridamole

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and Biochemical Parasitology 70 (1995) 59-69

&de&e

30s (pmol/pl

uptake cell water)

10 20 30s lnosine uptake (pmol/~I cell water)

C

inhibitor.

lnosine

Control [ 14C] lnosine Dipyridamole + Dipyridamole

Adenosine

Control [3H] Adenosine Dipyridamole + Dipyridamole

[14C]

[3H]

k

0

20

30s uptake

40

(pmol/pl

cell water)

Fig. 5 Inhibition of [3H]adenosine or [r4C]inosine transport by either labeled or unlabeled substrates. Transport of 20 /.LM L3H]adenosine (A) or [t4C]inosine (B) in the presence of unlabeled substrates (1.6 mM) measured at 30 s. Data illustrate single experiments representative of 4 and 2 independent experiments, respectively. Transport of 20 PM [3H]adenosine and 20 /.LM [‘4C]inosine (C) measured in the absence or presence of the other labeled substrate (20 PM) and in the presence or absence of dipyridamole (10 PM). Data show a single experiment representative of two separate experiments.

was minimal, and adenosine

although the effect of dipyridamole was additive.

4. Discussion We have identified an adenosine transporter in the plasma membrane of T. gondii. Transport of adeno-

sine is non-concentrative and Na+ independent, and is approx. 20-fold more sensitive to dipyridamole (IC,, approx. 0.7 PM) than to NBTI (IC,, approx. 15 PM). Since adenosine is the preferred purine substrate for salvage by T. gondii, this transporter may play a crucial role in the intracellular survival and replication of the parasite.

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Although studies of nucleoside transport processes in protozoan parasites are limited, most protozoa appear to have at least two separate nucleoside transport pathways. Experiments in Leishmania donovani [22,23], Trypanosoma cruzi [24], Trypanosoma brucei [25,26], Giardia intestinalis [27291, Giardia lamblia [30] and Trichomonas vaginalis [31] have identified 2 or 3 transport systems in the parasite plasma membrane which have restricted substrate specificity and distinct inhibitor profiles. These appear to be different from the broad specificity transporters of mammalian cells that are inhibited by both NBTI and dipyridamole. These protozoan transport systems vary in dipyridamole sensitivity but are uniformly resistant to inhibition by NBTI at concentrations < 10 PM. Our results are most simply explained by a single transporter for adenosine which also transports inosine, hypoxanthine and adenine. In this model, the minor inhibition by dipyridamole of inosine, hypoxanthine and adenine transport may reflect the presence of one or more separate dipyridamole resistant transporters for these substrates. Alternately, separate sites for adenosine and for other substrates are present on the same transporter, as suggested for the T. brucei P2 transporter [26] and for both Trichomonas uaginalis nucleoside transporters [31]. Given the ability of inosine, hypoxanthine and adenine to competitively inhibit adenosine transport, this latter possibility seems unlikely, since inhibition of substrate metabolism should not occur with adenine. Limited studies are available on nucleoside transport in Apicomplexan parasites. Gero et al. [32,33] identified a nucleoside permeation route in Plasmodium falciparum-infected erythrocytes which was insensitive to NBTI (10 PM), nitrobenzylthioguanosine, dilazep and dipyridamole. Treatment of Plasmodia-infected erythrocytes with the oxidizing agent diamide had no effect on adenosine transport, but did block the induced adenosine transport observed in Babes& bouis-infected bovine erythrocytes [34]. Potential transporters in the parasite plasma membrane were not investigated in these studies. Adenosine is rapidly converted to nucleotide after transport into tachyzoites of T. gondii. Since the parasite has an active and abundant adenosine kinase [7], and the majority of rapid nucleotide formation in the wild-type RH strain is in the form of adenine

67

nucleotides, direct conversion of adenosine to AMP, ADP and ATP by adenosine kinase is likely. Nonetheless, adenosine kinase is not essential for parasite viability since a mutant strain completely deficient in this enzyme has been isolated previously [8,15] and was used in the studies reported here. The kinetics of nucleotide formation after adenosine transport in the adenosine kinase deficient mutant were slower than in the wild-type RH strain. We will show elsewhere that the transported adenosine in the adenosine kinase deficient strain is rapidly converted to inosine and hypoxanthine, and subsequently to nucleotide. The calculated values for the apparent K, and Vmax varied significantly depending upon whether only values for unmetabolized substrate were used. This variation in K, and V,,, calculations depends upon the extent of substrate metabolism and illustrates the shortcomings of determining kinetic parameters for transport under circumstances where substantial metabolism of the labeled substrate is present. In most previously published studies in which the form of the transported label was studied, rapid metabolism occurred, even at extremely short uptake intervals [22,31]. Identification of an adenosine transporter in the plasma membrane of T. gondii tachyzoites and previous studies showing that adenosine is the most efficiently salvaged purine in vitro suggest but does not prove that adenosine could serve as a central purine source for the parasite in vivo. This parasite resides intracellularly within a unique vacuole which is isolated from membrane traffic [14]. Furthermore, mammalian host cell adenosine concentrations are < 1 FM., much lower than the K, suggested for mammalian nucleoside transporters [ 1 l] or that found for this parasite. We propose that the ultimate source of parasite adenosine is host cell ATP (present at 4-6 mM concentration in the host-cell cytosol). We speculate that ATP entering the vacuolar space through recently-identified pores in the parasitophorous vacuole membrane [35], is hydrolyzed to monophosphate form by a parasite protein nucleoside triphosphate hydrolase [36,37] discharged into the vacuole from parasite secretory organelles where it had been localized [37,38]. The resultant AMP is further hydrolyzed to adenosine by the ubiquitous plasma membrane marker enzyme 5’ nucleotidase

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[39] previously identified in other protozoans [40]. The presence of 5’ nucleotidase in T. gondii could potentially account for the reported marginally more rapid salvage of AMP compared to adenosine [6]. This novel extra-parasite intravacuolar nucleoside salvage pathway terminating with the parasite plasma membrane nucleoside transporter presents a new array of targets for anti-toxoplasma drug development.

Acknowledgments This work was supported by Public Health Service Grant UOl AI 31808 from the National Institutes of Allergy and Infectious Diseases (K.A.J.), by the Egyptian Mission (M.A.A.), by fellowships to J.C.S. from Miles Pharmaceuticals and the American Philosophical Society (Daland Fellowship) and American Cancer Society Grant CH67 (R.E.H.).

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