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ORIGINAL PAPER
Jose
´
Roberto Meyer-Fernandes Æ Jorge Saad-Nehme
Carlos E. Peres-Sampaio Æ Rodrigo Belmont-Firpo
Danielle F. R. Bisaggio Æ Luciana C. do Couto
Andre
´
Luı
´
z Fonseca de Souza Æ Angela H. S. C. Lopes
Thais Souto-Padro
´
n
A Mg-dependent ecto-ATPase is increased in the infective stages
of
Trypanosoma cruzi
Received: 1 December 2003 / Accepted: 4 December 2003 / Published online: 2 April 2004
Springer-Verlag 2004
Abstract In this work, we describe the ability of living
epimastigotes of Trypanosoma cruzi to hydrolyze extra-
cellular ATP. In these intact parasit es, there was a low
level of ATP hydrolysis in the absence of any divalent
metal (2.42±0.31 nmol Pi/h·10
8
cells). ATP hydrolysis
was stimulated by MgCl
2
, and the Mg-dependent ecto-
ATPase activity was 27.15±2.91 nmol Pi/h·10
8
cells.
The addition of MgCl
2
to the extracellular medium in-
creased the ecto-ATPase activity in a dose-dependent
manner. This stim ulatory activity was also observed
when MgCl
2
was replaced by MnCl
2
, but not by CaCl
2
or SrCl
2
. The apparent K
m
for Mg-ATP
2
was
0.61 mM, and free Mg
2+
did not increase the ecto-
ATPase activity. This ecto-ATPase activity was insen-
sitive to the inhibitors of other ATPase and phosphatase
activities. To confirm that this Mg-dependent ATPase
was an ecto-ATPase, we used an impermeant inhibitor,
DIDS (4, 4¢.diisothiocyanostylbene 2¢-2¢-disulfonic acid)
as well as suramin, an antagonist of P
2
purinoreceptors
and inhibitor of some ecto-ATPases. These two reagents
inhibited the Mg
2+
-dependent ATPase activity in a
dose-dependent manner. A comparison among the
Mg
2+
-ecto-ATPase activities of the three forms of
T. cruzi showed that the noninfective epima stigotes were
less efficient at hydrolyzing ATP than the infective try-
pomastigote and amastigote stages.
Keywords Trypanosoma cruzi Æ Ecto-ATPase Æ
Virulence
Introduction
Trypanosoma cruzi, the etiological agent of Chagas’
disease, is a parasitic protozoan with a complex life cycle
involving morphologically and functionally different
stages that enable these parasites to adapt to a variety of
conditions imposed by their insect vectors and mam-
malian hosts (Tanowitz et al. 1992).
Surface membrane interactions between parasites and
their host cells are of critical impo rtance for the survival
of the parasites, from both the immunological and
physiological viewpoints (Alexander and Russel 1992;
Martiny et al. 1996, 1999). Parasite membrane compo-
nents may play a role in the uptake of these organisms
by mammalian macrophages (Handman and Goding
1986; Russel and Wilhem 1986; Alexander and Russel
1992; Martiny et al. 1999). Plasma membranes contain
enzymes whose active sites face the external medium
rather than the cytoplasm. The activities of these en-
zymes, referred to as ecto-enzymes, can be measured
using inta ct cells (Furuya et al. 1998; Meyer-Fernandes
2002). Two examples are the trans-sialidase (Ming et al.
1993) and protein tyrosine phosphatase (Zhong et al.
1998) of T. cruzi. Different functions in host cell infec-
tion have been attributed to these enzymes. For exam-
ple, roles for the trans-sialidase (Ming et al. 1993) and
protein tyrosine phosphatase (Zhong et al. 1998) of
T. cruzi in the invasion process have been suggested.
Cell membrane ecto-ATPases are integral membrane
glycoproteins that are millimolar divalent, cation-
dependent, low specifity enzymes that hydrolyze all
nucleoside triphosphates (Plesner 1995; Kirley 1997;
J. R. Meyer-Fernandes (&) Æ J. Saad-Nehme
C. E. Peres-Sampaio Æ R. Belmont-Firpo
A. L. Fonseca de Souza
Departamento de Bioquı
´
mica Me
´
dica,
Instituto de Cieˆ ncias Biome
´
dicas,
Universidade Federal do Rio de Janeiro, CCS,
Bloco H, Cidade Universita
´
ria, Ilha do Funda
˜
o,
21541-590 Rio de Janeiro, RJ, Brazil
E-mail: meyer@bioqmed.ufrj.br
Tel.: +55-21-5904548
Fax: +55-21-2708647
D. F. R. Bisaggio Æ L. C. do Couto Æ A. H. S. C. Lopes
T. Souto-Padro
´
n
Instituto de Microbiologia Prof. Paulo de Go
´
es CCS,
Universidade Federal do Rio de Janeiro, Bloco I, CCS,
Cidade Universita
´
ria, , Ilha do Funda
˜
o,
21541-590 Rio de Janeiro, RJ, Brazil
Parasitol Res (2004) 93: 41–50
DOI 10.1007/s00436-003-1066-4
Meyer-Fernandes et al. 1997). The identity and function
of ecto-ATPases have been reviewed and the name ‘‘E-
type ATPas es’’ was proposed for these enzymes (Plesner
1995). Their physiological role is still unknown. How-
ever, several hypotheses have been suggested, such as:
(1) protection from the cytolytic effects of extracellular
ATP (Filippini et al. 1990; Zanovello et al. 1990; Stein-
berg and Di Virgilio 1991), (2) regulation of ectokinase
substrate concentration (Plesner 1995), (3) termination
of purinergic signaling (Weisman et al. 1996; Westfall
et al. 1997), (4) involvement in signal transduction
(Margolis et al. 1990; Dubyak and El-Moatassim 1993;
Yagi et al. 1994), and (5) involve ment in cellular adhe-
sion (Aurivillus et al. 1990; Cheung et al. 1993;
Dzhandzhugazyan and Bock 1993; Stout et al. 1995;
Kirley 1997).
Here, we show the presence of a Mg
2+
-dependent
ecto-ATPase activity on the cell surface of living
T. cruzi, and characterize the properties of this enzyme.
We also compare the ATP hydrolysis catalyzed by the
noninfective epimastigotes and the infective trypomas-
tigote and amastigote stages.
Materials and methods
Culture methods
The Y strain of T. cruzi was used throughout this study. Epimas-
tigote forms were maintained at 28C under constant shaking (120
oscillations/min) in Warren medium (Warren 1960), supplemented
with 10% fetal calf serum and used on the first to third days of
cultivation. Parasites were collected by centrifugation, washed
twice and kept in 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM
D-glu-
cose, 50.0 mM Hepes-Tris buffer, pH 7.2. Cell growth was esti-
mated daily by counting the number of parasites in a Neubauer
chamber. For the isolation of the trypomastigote and amastigote
forms, LLCMK2 cells were infected with tissue culture trypom-
astigotes. After 5–6 days, the supernatant was collected, centri-
fuged at 500 g for 5 min, and allowed to stand at 37C for 30 min.
During this period, the trypomastigotes in the pellet moved into the
supernatant. The amastigote forms stayed in the pellet. Over 95%
of the pellet was made up of amastigotes and over 95% of the
supernatant was composed by trypomastigotes. These trypom-
astigotes were then collected and centrifuged at 1,000 g for 10 min.
The contamination of trypomastigotes with amastigotes and
intermediate forms or of amastigotes with trypomastigotes and
intermediate forms was always less than 5%. Cellular viability was
assessed, before and after incubation, by motility and trypan blue
exclusion. For trypan staining, the cells were incubated in the
presence of 0.01% trypan blue for 10 min in the buffer used in each
experiment (Dutra et al. 2001a). The viability was not affected
under the conditions employed here.
Ecto-ATPase activity measurements
Intact cells were incubated for 1 h at 30C in 0.5 ml of a mixture
containing, unless otherwise specified, 116.0 mM NaCl, 5.4 mM
KCl, 5.5 mM
D-glucose, 50.0 mM Hepes-Tris buffer, pH 7.2,
5.0 mM ATP, and 3.0·10
7
cells/ml, in the absence or presence of
5.0 mM MgCl
2
. The Mg
2+
-dependent ecto-ATPase activity was
calculated from the total activity, measured in the presence of
5 mM MgCl
2
, minus the basal activity, measured in the absence of
MgCl
2
. The ATPase activity was determined by measuring the
hydrolysis of [c-
32
P]ATP (10
4
Bq/nmol ATP) (Dos Passos Lemos
et al. 2000). The experiments were started by the addition of living
cells and terminated by the addition of 1.0 ml of a cold mixture
containing 0.2 g charcoal in 1.0 M HCl. The tubes were then
centrifuged at 1,500 g for 10 min at 4C. Aliquots (0.5 ml) of the
supernatants containing the released
32
Pi were transferred to scin-
tillation vials containing 9.0 ml of scintillation fluid. The ATPase
activity was calculated by subtracting the nonspecific ATP hydro-
lysis measured in the absence of cells. The ATP hydrolysis was
linear with time under the assay conditions used and was propor-
tional to the cell number. In the experiments in which other nu-
cleotides were used, the hydrolytic activities measured under the
same conditions described above were assayed spectrophotomet-
rically by measuring the release of P
i
from the nucleotides (Lowry
and Lopes 1946). The values obtained for ATPase activities mea-
sured using both methods (spectrophotometric and radioactive)
were exactly the same. In the experiments in which high concen-
trations of Mn
2+
,Ca
2+
and Sr
2+
were tested, possible precipitates
were checked as previously described (Meyer-Fernandes and Vie-
yra 1988). Under the conditions employed in the reaction medium
containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl,
5.5 mM
D-glucose and 5 mM ATP, no phosphate precipitates were
observed in the presence of these cations.
Phosphatase measurements
In addition to the measurements of ecto-ATPase activity, the ecto-
p-nitrophenylphosphatase activity was determined in the same
medium as that for ATP hydrolysis except that ATP was replaced
by 5.0 mM p-nitrophenylphosphate (p-NPP). The reaction was
determined spectrophotometrically at 425 nm using an extinction
coefficient of 14.3·10
3
M
1
cm
1
(Meyer-Fernandes et al. 1999).
Statistical analysis
All experiments were performed in triplicate, with similar results
obtained in at least three separate cell suspensions. Apparent K
m
and V
max
values were calculated using an iterative nonlinear
regression analysis of the data to the Michaelis-Menten equation
(Saad-Nehme et al. 1997). Statistical significance was determined
by Student’s t-test. Significance was considered as P<0.05.
Chemicals
All reagents were purchased from Merck (Darmstadt, Germany) or
Sigma (St. Louis, Mo.). [c
32
P] ATP was prepared as described by
Glynn and Chappell (1946). Distilled water was deionized using a
MilliQ system of resins (Millipore, Bedford, Mass.) and was used in
the preparation of all solutions. Concentrations of free and com-
plexed species (Mg
2+
, ATP
4
and MgATP
2
) at equilibrium were
calculated by using an iterative computer program that was mod-
ified (Sorenson et al. 1986) from that described by Fabiato and
Fabiato (1979).
Results
T. cruzi epimastigotes, whose viability was assessed
before and after the reactions by motility and trypan
blue exclusion, presented low ATP hydrolysis
(2.42±0.31 nmol Pi/h·10
8
cells) in the absence of any
divalent metal (1 mM EDTA). At pH 7.2, the addition
of 5 mM MgCl
2
stimulated ATP hydrolysis, and Mg
2+
-
dependent ecto-ATPase activity [difference between total
(measured in the presence of 5 mM MgCl
2
) minus basal
ecto-ATPase activity (measured in the presence of 1 mM
42
EDTA] in these parasites hydrolyzed ATP at
27.15±2.91 nmol Pi/h·10
8
cells. The time course of
ATP hydrolysis by the T. cruzi Mg
2+
-dependent ecto-
ATPase was linear for at least 90 min (Fig. 1A). Simi-
larly, in assays to determine the influence of cell density,
the Mg
2+
-dependent activity measured over 60 min was
linear over a nearly sixfold range of cell densities
(Fig. 1B). In order to check the possibility that the ob-
served ATP hydrolysis was the result of secreted soluble
enzymes, as observed for other parasites (Bermudes
et al. 1994; Smith et al. 1997), we prepared a reaction
mixture with cells that were incubated in the absence of
ATP. Subsequently, the suspension was centrifuged to
remove cells and the supernatant was checked for AT-
Pase activity. This supernatant failed to show ATP
hydrolysis either in the absence or presence of MgCl
2
(data not shown). These data also rules out the possi-
bility that the ATPase activity could be from lysed
T. cruzi cells.
A possible contribution to ATP hydrolysis promoted
by acid phosphatase activity present in several species of
the Trypanosomatidae (Meyer-Fernandes et al. 1999;
Dutra et al. 2000) was examined by measuring the pH
dependence of both activities. In the pH range from 6.5
to 8.5, in which the cells were alive throughout the
reaction, the phosphatase activity dec reased concomi-
tantly with the pH increase (Fig. 2A). On the other
hand, the Mg
2+
-dependent ATPase activity was not
affected by pH variation (Fig. 2B). Several inhibitors of
phosphatase and ATPase activities different from the
ecto-ATPase described in this work were tested in order
to exclude the possibility that the ATP hydrolysis was
due to the mentioned enzymes. Table 1 shows that so-
dium fluoride (NaF) and ammonium molybdate, two
potent inhibitors of aci d phosphatase (Fernandes et al.
1997; Dutra et al. 1998, 2001b), had no effect on ATPase
activity. Levamizole, a specific inhibitor of alkaline
phosphatases (Van Belle 1976), also failed to inhibit the
ATP hydrolysis catalyzed by intact T. cruzi. In addition,
the lack of response to p-nitrophenylphosphate (p-NPP),
a substrate for phosphatase activity (Table 1), indicates
that this enzyme did not contribute to the observed ATP
hydrolysis. The Mg-dependent ATPase activ ity was
insensitive to oligomycin and sodium azide, two inhibi-
tors of mitochondrial Mg-ATPase (Meyer-Fernandes
et al. 1997); bafilomycin A
1
, a V-ATPase inhibitor
(Bowman et al. 1988); ouabain, a Na
+
/K
+
-ATPase
inhibitor (Caruso-Neves et al. 1998a); furosemide, a
Na
+
-ATPase inhibitor (C aruso-Neves et al. 1998b); as
Fig. 1 A Time course and B cell density dependence of the Mg
2+
-
dependent ecto-ATPase activity of intact cells of Trypanosoma
cruzi. Cells were incubated for different periods of time (A) or for
1h(B)at30C, in a reaction medium containing 50 mM Hepes-
Tris buffer, pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM
D-glucose, and 5 mM Tris-ATP (c-
32
P) ATP (sp act=10
4
Bq/nmol
ATP) in the absence or in the presence of 5.0 mM MgCl
2
. The
Mg
2+
-dependent ecto-ATPase activity was calculated from
the total activity, measured in the presence of 5 mM MgCl
2
, minus
the basal activity, measured in the absence of MgCl
2
. Data are
means±SE of three determinations using different cell suspensions
Fig. 2 The effect of pH on the ecto-phosphatase and ecto-ATPase
activities of intact cells of T. cruzi. Cells were incubated for 1 h at
30C in a reaction medium containing 116 mM NaCl, 5.4 mM
KCl, 5.5 mM D-glucose, 5.0 mM MgCl
2
, 3.0·10
7
cells/ml, 50 mM
Hepes-Tris buffer, adjusted to pH values between 6.5 and 8.5 with
HCl and Tris, in the presence of A 5mMp-NPP, or B 5 mM ATP.
In this pH range, the cells were viable throughout the course of the
experiments. Data are means±SE of three determinations using
different cell suspensions
43
well as to vanadate, which is a potent inhibitor of
P-ATPases (Sodre
´
et al. 2000).
Since we used intact parasites for measuring the en-
zyme activities in all of the experiments done in this
study, it is likely that the Mg
2+
-dependent ATPase
activity described is due to an ecto-enzyme. To confirm
this, we applied the criterion that an authentic ectoen-
zyme should be inhibited by an extracellular impermeant
inhibitor (Knowles 1988; Barbacci et al. 1996; Meyer-
Fernandes et al. 1997) such as 4,4¢-diisothiocyanostyl-
bene 2¢-2¢-disulfonic acid (DIDS) (Knowles 1988;
Barbacci et al. 1996; Meyer-Fernandes et al. 1997), and
possibly by an ecto-ATPase inhibitor, such as suramin,
which is also an antagonist of P
2
-purinergic receptors
(Hourani and Chown 1989; Ziganshin et al. 1995). As
shown in Fig. 3, the Mg
2+
-dependent ecto-ATPase
activity was inhibited by DIDS (panel A) and suramin
(panel B), both in a dose- dependent manner. The inhi-
bition promoted by both DIDS and suramin was
attenuated when the cells were incubated in the presence
of a high concentration of ATP (Fig. 3A and B, open
circles). We have previously shown that DIDS inhibits
T. cruzi epimastigote cell growth (Bernardes et al. 2000),
and that this inhibition is supposed to be related to ecto-
enzymes involved in cell proliferation (Berreˆ do-Pinho
et al. 2001). ATP and other nucleotides stimulate the
proliferation of different cell types, while the inhibition
of the ecto-ATPase, 5¢ nucleotidase and alkaline phos-
phatase moderate the stimulatory effect of ATP
(Lemmens et al. 1996). Figure 4 shows that the Mg
2+
-
dependent ATPase activity decreased during the time
course of cell growth. The Mg
2+
-dependent ATPase
activity is sixfold higher on the second day than on the
sixth day (Fig. 4).
Mg
2+
is an important extracellular signal in the
regulator of Salmonella virulence (Ve
ˇ
scovi et al. 1996).
Table 1 Influence of various agents on Mg-dependent ecto-ATPase
activity of T. cruzi. ATPase activity was measured at pH 7.2. It is
expressed as a percentage of that measured under control condi-
tions, i.e., without other additions. The ATPase (24.8±3.5 nmol
Pi/h·10
8
cells) activity was taken as 100%. The standard errors
were calculated from the absolute activity values of three experi-
ments with different cell suspensions and converted to a percentage
of the control values. An unpaired t-test showed, in all cases, that
there were no statistical differences between compounds. For rel-
ative activity, the final concentrations of the different agents were
the highest for which there was no alteration in the parasite
integrity
Additions Relative activity
Control 100.0±13.4
Levamizole (1.0 mM) 96.7±12.3
Vanadate (1.0 mM) 102.7±13.0
Tartrate (10.0 mM) 101.3±14.2
NaF (10.0 mM) 109.9±10.2
Molybdate (1.0 mM) 99.8±11.6
p-NPP (10 mM) 103.8±9.5
AMP (10 mM) 107.1±11.4
Bafilomycin A
1
(10.0 lM) 96.2±0-5
Azide (10.0 mM) 89.7±9.4
Oligomycin (10.0 lg/ml) 113.4±14.1
Ouabain (1.0 mM) 90.6±10.8
Furosemide (1.0 mM) 96.2±9.7
Dipyridamole (10.0 lM) 91.6±11.5
Fig. 3 The effect of increasing concentrations of DIDS and
suramin on the Mg
2+
-dependent ecto-ATPase activity of intact
cells of T. cruzi. Cells were incubated for 1 h at 30C in the same
reaction medium (final volume 0.5 ml) as that described in the
legend of Fig. 1, in the absence or in the presence of 5.0 mM MgCl
2
in the presence of 1 mM ATP (d) or 10 mM ATP (s) with A
increasing concentrations of DIDS, or B suramin. The Mg
2+
-
dependent ecto-ATPase activity was calculated from the total
activity, measured in the presence of 5 mM MgCl
2
, minus the basal
activity, measured in the absence of MgCl
2
. Data are means±SE of
three determinations using different cell suspensions
Fig. 4 The growth curve (d) and ecto-ATPase activity (s) of intact
cell of T. cruzi. Parasites were cultivated for 6 days. Values shown
are the mean of triplicate determinations from three different
experiments
44
We ha ve also shown that a pathogenic Entamoeba his-
tolytica strain has a much higher Mg
2+
-dependent ecto-
ATPase activity than the noninvasive E. histolytica or
the free-living E. moshkovskii (Barros et al. 2000). The
addition of MgCl
2
to the extracellular medium stimu-
lated the ecto-ATPase activity of T. cruzi in a dose-
dependent manner (Fig. 5, open circles). At 5 mM ATP,
half of the max imum stimulation of ATP hydrolysis was
obtained in the presence of 0.72 mM MgCl
2
. The addi-
tion of MnCl
2
to the extracellular medium also pro-
moted an increase in this ecto-AT Pase activity in a dose
dependent manner (Fig. 5, closed circles). The stimula-
tion observed by Mg
2+
and Mn
2+
was not observed
when these cations were replaced by Ca
2+
(Fig. 5, open
squares) or Sr
2+
(Fig. 5, closed squares). Under the
conditions employed, in the reaction medium containing
50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl,
5.5 mM D-glucose and 5 mM ATP in the absence of any
divalent cation, the concentration of ATP
4
was
2.7 mM. In the presence of 10 mM CaCl
2
, the concen-
trations of ATP
4
and Ca-ATP
2
were 0.1 and 4.7 mM,
respectively. The changes in ATP
4
and Ca-ATP
2
levels had no effect on ATPase activity (Fig. 5, open
squares). Th ese data indicate that Mg-ATP
2
is the
substrate for this enzyme. The apparent K
m
for Mg-
ATP
2
was 0.61 mM (Fig. 6) and free Mg
2+
did not
increase the ecto-ATPase activity (Fig. 6, inset). We
analyzed the specificity of this Mg
2+
-dependent ecto-
ATPase activity for other nucleotides. Table 2 shows
that ATP was the best substrate for this enzyme and that
ecto-ATPase hydrolyzed under nucleoside 5¢-triphos-
phates ITP, CTP, GTP and UTP at high rates. ADP was
not recognized as a substrate, indicating that it is an
ecto-ATPase (Heine et al. 1999) and not an ecto-ATP
diphosphohydrolase, described in other cell types
(Handa and Guidotti 1996; Wang and Guidotti 1996;
Meyer-Fernandes et al. 2000). Another possible
Fig. 5 The influence of different cation concentrations on the ecto-
ATPase activities of intact cells of T. cruzi. Cells were incubated for
1 h at 30C in the same reaction medium (final volume: 0.5 ml) as
that described in the legend of Fig. 1, with the addition of
increasing concentrations of Mg
2+
(s), Mn
2+
(d), Ca
2+
(h)or
SR
2+
(n). Data are means±SE of three determinations using
different cell suspensions
Fig. 6 Dependence on Mg-ATP
2
concentrations of the ecto-
ATPase activity of intact T. cruzi. cells The cells were incubated for
1 h at 30C in the same reaction medium (final volume 0.5 ml) as
that described in the legend of Fig. 1, which corresponds to Mg-
ATP
2
concentrations varying as shown on the abscissa. The curve
represents the fit of experimental data by nonlinear regression using
the Michaelis-Menten equation. Inset: the effects of free Mg
2+
on
the ecto-ATPase activity. Cells were incubated for 5 min at 30Cin
the same reaction medium (final volume 0.5 ml) as that described in
the legend of Fig. 1, with the addition of 1 mM EDTA and 10 lM
Mg-ATP
2
which corresponds to Mg
2+
concentrations varying as
shown on the abscissa. The total amounts of ATP and MgCl
2
necessary to form the desired Mg-ATP
2
and free Mg
2+
concentrations were calculated as described. Data are means±SE
of three determinations using different cell suspensions
45
explanation for the ATP hydrolysis is that 5¢ nucleo-
tidase, an other enzyme present on the external surface of
T. cruzi (Fig. 7), could be responsible. However, as can
be seen in Fig. 7, the addition of MgCl
2
to the extra-
cellular medium stimulated only ecto-ATPase activity,
whereas no effect was observed on AMP hydrolysis or
on ADP and p-NPP hydrolysis. The lack of response to
ammonium molybdate (Table 1), a potent inhibitor of 5¢
nucleotidase (Gottlieb and Dwyer 1983), and to its
substrate, AMP (Table 1), indicate that this enzyme did
not contribute to the observed ATP hydrolysis. In
addition, suramin did not inhibit AMP hydrolysis or
ADP hydrolysis (data not shown). These data confirm
that the ATP hydrolysis stimulated by Mg
2+
is cata-
lyzed by an authentic Mg-dependent ecto-ATPase.
It is well known that trypanosomatids of the genus
Trypanosoma are unable to synthesize purines de novo,
and thus are dependent on exogenous sources of these
essential nutrients (De Koning et al. 2000; Berreˆ do-
Pinho et al. 2001). Extracellular ATP and its degrada-
tion products ADP, AMP and adenosine are normal
components of the extracellular milieu. Extracellular
nucleotides do not cross the cell memb rane, but rather
mediate their biological actions through specific recep-
tors on the cell surface where they are locally metabo-
lized by ecto-nucleotidases (El-Moatassim et al. 1992;
Dombrowski et al. 1998). The three different enzymatic
activities (ecto-ATPase, ecto-ADPase and ecto-5¢ nuce-
lotidase) present on the surface of T. cruzi (Fig. 7) might
sequentially dephosphorylate ATP to adenosine :
ATP fi ADP fi AMP fi adenosine, making adenosine
available to T. cruzi from nucleotides which, because of
their charge, are not permeable to the plasma mem-
brane. The physiological role of the ecto-ATPase is still
unknown, but a possible involvement in cellular adhe-
sion has been proposed (Dzhandzhugazyan and Bock
1993; Stout et al. 1995; Kirley 1997; Barros et al. 2000;
Berreˆ do-Pinho et al. 2001; Meyer-Fernandes 2002).
Recently, we have shown that the invasive form of E.
histolytica (Barros et al. 2000) and virulent promastig-
otes of Leishmania amazonensis (Berreˆ do-Pinho et al.
2001) have a much higher Mg-depe ndent ecto-ATPase
activity than the noninvasive form E. histolytica (Barros
et al. 2000) or the avirulent promastigotes of L. ama-
zonensis (Berreˆ do-Pinho et al. 2001), suggesting that this
enzyme could be considered a marker of pathogenesis
for these parasites. As shown in Fig. 8, the infective
stages (trypomastigotes and amastigotes) of T. cruzi
showed 20-fold more Mg-dependent ecto-ATPase
activity than noninfective epimastigotes. Galactose
exposed on the surface of human erythrocytes plays
an important role in the interaction of those cells with
T. cruzi (Silber et al. 2002). We have previously shown
that
D-galactose stimulates a Mg
2+
-dependent ecto-
ATPase activity of E. histolytica (Barros et al. 2000).
Accordingly, the ecto-ATPase of T. cruzi was stimulated
by
D-galactose in a dose-dependent manner (Fig. 9).
Discussion
This paper reports the presence of an Mg-dependent
ecto-ATPase on the external surface of T. cruzi. Cellular
integrity and viability were assessed, before and after the
reactions, by mobility and trypan blue exclusion (Dutra
et al. 2001a). The integrity of the cells was not affected
by any of the conditions used in the assays. The external
location of the ATP-hydrolyzing site is supported by its
sensitivity to the imperm eant inhibitor DIDS (Fig. 3A)
(Knowles 1988; Barbacci et al. 1996; Meyer-Fernandes
et al. 1997), and to suramin (Fig. 3B), which is a non-
competitive inhibitor of ecto-ATPases and an antagonist
Table 2 Substrate specificity of Mg-dependent ecto-nucleotidase
activity of T. cruzi. Ecto-nucleotidase activity was measured at
30C in medium containing the nucleotides listed (5 mM), 50 mM
Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM
D-glucose and
3.0·10
7
cells/ml in the absence or in the presence of 5.0 mM MgCl
2
.
The Mg
2+
-dependent ATPase (26.2±3.3 nmol P
i
/h·10
8
cells)
activity (difference between total, measured in the presence of
5 mM MgCl
2
, minus basal ecto-ATPase activity, measured in the
absence of MgCl
2
) was taken as 100%. The standard errors were
calculated from the absolute activity values of three experiments
with different cell suspensions and converted to a percentage of the
control value. In these experiments, Pi release from all nucleotides,
including ATP, was measured using a spectrophotometric assay
Nucleotides Relative activity %
ATP 100.0±9.1
UTP 87.6±8.1
CTP 81.5±11.6
GTP 78.5±8.4
ITP 71.1±9.3
ADP 0
Fig. 7 The influence of MgCl
2
on the ecto-phosphohydrolase
activities of intact T. cruzi cells. The cells were incubated for 1 h
at 30C in the same reaction medium (final volume: 0.5 ml) as that
described in the legend of Fig. 1, in the presence of 5 mM of either
ATP, ADP, 5¢AMP or p-NPP. Black bars show total activity,
measured in the presence of 5 mM MgCl
2
. Blank bars show basal
activity, measured in the absence of MgCl
2
. In these experiments
ATP hydrolysis was measured using the same spectrophotometric
assay for P
i
release as that used for the other nucleotides. Data are
means±SE of three determinations using different cell suspensions
46
of P
2
purinoreceptors, which mediate the physiological
functions of extracellular ATP (Hourani and Chown
1989; Ziganshin et al. 1995). Also, a battery of inhibitors
for other ATPases that have intracellular ATP binding
sites showed no effect of the ecto-ATPase activ ity (Ta-
ble 1). The Mg-dependent ATPase activity reported in
this work could not be attributed to a mitochondrial
ATPase, since this activity wa s insensitive to oligomycin
and sodium azide (Table 1), two known F-type ATPase
inhibitors (Meyer-Fernandes et al. 1997). Since the Mg-
dependent ecto-ATPase described here did not respond
to vanadate (Table 1), the possibility that this activity
was due to a P-ATPase present on the surface of the
plasma membrane was discarded. For these reasons, we
assign an ectolocalization to the Mg-dependent ATPase
activity described here (Plesner 1995; Me yer-Fernandes
et al. 1997, 2000; Barros et al. 2000; Berreˆ do-Pinho et al.
2001). This ATP hydrolysis could not be due to phos-
phatase activity present on the external surface of the
T. cruzi membrane, because, as shown in Table 1, potent
inhibitors for phosphatase activities were not capable of
modifying the Mg-dependent ecto-ATPase activity. The
Mg-dependent ecto-ATPase activity described here
could not be attributed to a 5¢ nucleotidase, since the
AMP hydrolysis was not stimulated by Mg
2+
(Fig. 7);
in addition, ammonium molybdate, a potent inhibitor of
5¢ nucleotidase (Gottlieb and Dwyer 1983), did not in-
hibit the Mg-dependent ecto-ATPase activity (Table 1).
The addition of MgCl
2
(Fig. 5, open circles) and
MnCl
2
(Fig. 5, closed circles) to the extracellular med-
ium stimulated the ecto-ATPase activity in a dose-
dependent manner. Most of the ecto-ATPases are Mg
2+
or Ca
2+
stimulated (Plesner 1995), however, this enzyme
was not stimulated by CaCl
2
(Fig. 5, open squares).
These data suggest that CaATP
2
is a substrate for this
enzyme, as it has been shown for the ecto-A TPase
present in Leishmania tropica (Meyer-Fernandes et al.
1997) and L. amazonensis (Berreˆ do-Pinho et al. 2001).
The substrate for the enzyme described here is the
complex MgATP
2
(K
m
=0.61 mM, Fig. 6), and free
Mg
2+
was not able to increase the enzyme activity
(Fig. 6, inset). Accordingly, the ecto-ATPase from
L. amazonensis (Berreˆ do-Pinho et al. 2001) and the
nucleoside triphosphate hydrolase from Toxoplasma
gondi (Asai et al. 1995) gave similar results. The Mg
2+
-
independent and the Mg
2+
-dependent ecto-ATPase
activities present in E. histolytica have a high specificity
for ATP, being much less active toward other nucleoside
triphosphate substrates (Barros et al. 2000). The Mg
2+
-
dependent ecto-ATPase was present in T. cruzi hydro-
lyses ATP, ITP, GTP, CTP and UTP at high rates
(Table 2). This enzyme did not recognize ADP as a
substrate, although ADP can be hydrolyzed by T. cruzi
in the absence of MgCl
2
(Fig. 7). The nucleoside tri-
phosphate hydrolyse (NTPase) purified from T. gondii is
not a single enzyme, but a mixture of two isozymes,
termed NTPase I and NTPase II, and a primary differ-
ence between these isozymes is that NTPase II hydro-
lyzes nucleoside triphosphate and diphosphate
substrates at almost the same rates, whereas NTPase I is
almost exclusively limited to nucleoside triphosphate
hydrolysis (Asai et al. 1995). Recently it was show n that
avirulent T. gondii strains express only NTPase II,
whereas virulent strains express both NTPase I and
NTPase II (Nakaar et al. 1998).
T. cruzi , as well as L. amazonensis, are pathogens
which cannot synthesize purines de novo (De Koning
et al. 2000; Berreˆ do-Pinho et al. 2001). It has been
Fig. 8 Mg
2+
-dependent ecto-ATPase activity of different stages of
T. cruzi. Intact epimastigotes (blank bars), trypomastigotes (black
bars) and amastigotes (hatched bars) were obtained as described in
the Materials and methods. The cells were incubated for 1 h at
30C in the same reaction medium (final volume 0.5 ml) as that
described in the legend of Fig. 1, in the absence or in the presence
of 5.0 mM MgCl
2
. The Mg
2+
-dependent ecto-ATPase activity was
calculated from the total activity, measured in the presence of
5 mM MgCl
2
, minus the basal activity, measured in the absence of
MgCl
2
. Data are means±SE of three determinations using
different cell suspensions
Fig. 9 Dependence on D-galactose concentrations on the ecto-
ATPase activity of intact T. cruzi cells. The cells were incubated for
1 h at 30C in the same reaction medium (final volume 0.5 ml) as
that described in the legend of Fig. 1, which corresponds to
D-galactose concentrations varying as shown on the abscissa. Data
are means±SE of three determinations using different cell
suspensions
47
postulated that these ecto-ATPases could play a role in
the salvage of purines from the host cells in protozoan
parasites (Berreˆ do-Pinho et al. 2001; Meyer-Fernandes
2002). The ability of T. cruzi to hydrolyze ATP, ADP
and AMP (Fig. 7) might sequentially dephosphorylate
ATP to adenosine: ATP fi ADP fi AMP fi adeno-
sine, indicating that this enzyme in T. cruzi might play a
role in the salvage of purines from the extracellular
medium. The physiological functions of the ecto-ATP-
ases are not known, however, many functions have been
hypothesized, including roles in the termination of pu-
rinergic signaling, purine recycling and cellular adhesion
(Plesner 1995). Considering the fact that T. cruzi is
responsible for complicated infections and that virulent
strains of T. gondii express an isozyme (NTPase I) from
a new family of E-type ATPases that is not expressed by
avirulent T. gondii strains (Nakaar et al., 1998), and that
the invasive form of E. histolytica has much higher Mg-
dependent ecto-ATPase activity than the noninvasive
form (Barros et al. 2000), one could also speculate that
the presence of an Mg-dependent ecto-ATPase activity
in T. cruzi might also reflect some form of evasion of the
host defense mechanisms in the circulation. Taking into
account that ATP can be released by different kinds of
cells, such as neutrophils and endothelial cells (Fred-
holm 1997), and the role of these cells in the pathogen-
esis of Chagas’ disease (Chen et al. 2001; Petkova et al.
2001), we suggested that Mg
2+
-dependent ecto-ATPase
activity on T. cruzi could stimulate the adherence of
parasites to the host cell and protect them from neu-
trophil attack through releasing adenosine to inhibit
superoxide production. The results presented in Fig. 8
demonstrate that the infective stages (trypomastigotes
and amastigotes) of T. cruzi have much higher Mg-
dependent ecto-ATPase activity than noninfective
epimastigotes.
D-Galactose, a sugar moiety known to be
an important adhesion molecule between mammalian
host cells and T. cruzi (Silber et al. 2002), promoted 90%
of stimulation of this Mg-dependent ecto-ATPase
activity (Fig. 9). The presence of a 67-kDa galactose-
binding protein present of the surface of T. cruzi , which
is involved in the invasion of human erythrocytes by this
parasite, was recently demonstrated (Silber et al. 2002).
We do not what the relationship between this 67-kDa
galactose-binding protein and the Mg-dependent ecto-
ATPase activity stimulated by galactose described here
is. Further investigations will be necessary in order to
determine whether Mg-dependent ecto-ATPase is an
adhesion molecule in T. cruzi, which could be considered
as a pathogenesis marker for this parasite.
The recently identified family of ecto-nuceloside tri-
phosphate diphosphohydrolases (E-NTPDase family)
contains multiple members that differ in their substrate
specificities and cellular locations (Zimme rmann 1999).
Mammalian memb rane associated ecto-ATPDase is
homologous to human CD39, a lymphoid cell activation
antigen (Handa and Guidotti 1996) . Wang and Guidotti
(1996) discovered that CD39 has sequence homology
with a potato apyrase and that CD39 has apyrase
activity. This work led to the identification of a family of
ecto-ATPases that are related in sequence but vary in
their membrane topology and tissue distribution (Ples-
ner 1995; Zimmermann 1999; Goding 2000). Further
characterization of cloned members of proteins related
to CD39 suggested a unifying nomenclature. All mem-
bers of the CD39-ATP diphosphohydrolase family be-
long to the E-NTPDase family (Lemmens et al. 2000;
Zimmermann 2000). Elucidation of the primary se-
quence of T. cruzi ecto-ATPase will be required to
positively identify this enzyme as a member of this
family.
Acknowledgements We would like to acknowledge the excellent
technical assistance of Fabiano Ferreira Esteves. This work was
partially supported by grants from the Brazilian agencies Conselho
Nacional de Desenvolvimento Cientı
´
fico e Tecnolo
´
gico (CNPq),
Coordenac¸ a
˜
o de Aperfeic¸ oamento de Pessoal de Nı
´
vel Superior
(CAPES), Financiadora de Estudos e Projetos (FINEP), Fundac¸ a
˜
o
de Amparo a
`
Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Programa de Nu´ cleos de Exceleˆ ncia (PRONEX, grant 0885) and
FUJB/UFRJ.
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