From the
A nitric oxide synthase was partially purified from soluble
extracts of Trypanosoma cruzi epimastigote forms. The
conversion of L-arginine to citrulline by this enzyme activity
required NADPH and was blocked by EGTA. The reaction was activated by
Ca
In mammalian cells, L-arginine is metabolized to yield
nitric oxide (NO),
NO synthases are the enzymes responsible for the
conversion of L-arginine to NO and citrulline. These enzymes
require NADPH and possess binding sites for heme, tetrahydrobiopterin,
flavin adenine dinucleotide, and flavin adenine mononucleotide. Two
groups of isoforms are usually defined for these synthases:
constitutive and inducible. NO synthases of the first group, found in
endothelium and neurons, are regulated by agonist-induced elevation of
intracellular
Ca
In neural cells,
constitutive NO synthase is modulated by the activity of a L-glutamate receptor subtype specific for N-methyl-D-aspartate (NMDA). Receptors of this
subtype control the voltage-dependent uptake of
Ca
From an evolutionary
viewpoint, evidence indicates that the NO transduction signaling
pathway is operative only in higher eukaryotic organisms. The present
studies provide the first demonstration that this pathway is also
present in the lower eukaryotic organism Trypanosoma cruzi,
the ethiological agent of the Chagas' disease. The existence of
Ca
Cells were collected
by centrifugation at 1000
The soluble crude extract (25 ml) was
loaded onto a DEAE-cellulose column (1
The authenticity of the radioactive citrulline
formed during the reaction was ascertained by comigration with a
citrulline standard on a silica gel 60 plate developed with
CHCl
NO generation by epimastigote cultures
was monitored by the formation of NO
Addition of protease inhibitors to the crude extracts is absolutely
necessary to preserve NO synthase activity. The high proteolytic
activity, which is characteristic of these extracts, might explain the
5-fold difference in total activity observed between the two first
steps of purification ().
Under the assay conditions
described by Bredt and Snyder (16) for neural tissues,
conversion of [
The effect of excitatory amino acids
was also studied by monitoring the concentration of NO in the
incubation medium as accumulation of NO
The effects of amino acids and sodium nitroprusside on
cyclic GMP levels was studied in T. cruzi epimastigote cells.
As shown in , glutamate and NMDA were the most potent
agents in increasing such levels. Sodium nitroprusside had a slightly
smaller effect, while L-arginine alone was much less active.
The binding of [
Results reported in this article show that T. cruzi epimastigotes have a NO synthase activity similar to that
previously described for mammalian endothelium and nervous
tissue(6, 7, 8, 9) . Enzymes from these
tissues show Ca
Some of
the excitatory amino acids, well known to affect the conversion of L-arginine to citrulline and NO in neural tissue, also
influence the T. cruzi NO synthase in vivo.
Remarkable stimulatory effects of L-glutamate and NMDA could
be observed in epimastigote cells, suggesting that T. cruzi epimastigotes have L-glutamate (NMDA) receptors of the
type described for nervous tissue(13) . As occurs in neural
cells, T. cruzi NMDA receptors should be major entities
controlling cytosolic Ca
A well known
feature of neural NMDA receptors is also found in T. cruzi epimastigote membrane receptors. This is the
[
On the other hand, the failure to detect
specific binding at the level of the L-glutamate-NMDA site in
the receptor may be attributable to the usually very low affinity of
this site for ligands such as L-glutamate, NMDA, CPP, or AP-5
(24).
The NO pathway, controlled through NMDA receptors in neural
cells, possesses a heme-containing soluble guanylyl cyclase as its
effector. This enzyme can be activated by sodium nitroprusside through
the generation of NO(3, 4) . This also seems to be the
case of T. cruzi epimastigotes, since NMDA and excitatory
amino acids such as L-glutamate, which activate NO synthase in vivo, also increase intracellular levels of cyclic GMP in
epimastigote cells.
It is known that NO generated by macrophages is
cytostatic or cytotoxic for a variety of pathogens, including Trypanosoma brucei and T.
cruzi.(25, 26) . Moreover T. cruzi infection in mice increases the capacity of splenic cells to
produce NO(27) . Obviously, the relationship between the two
NO-generating systems in the parasite and the mammalian cell remains
unknown.
Finally, it is rather surprising that a neural control
mechanism such as the long-term potentiation involved in memory (28) has in Trypanosomatidae such an old evolutionary
precedent. Both cases involve NMDA receptors, a
Ca
NO synthase activity was assayed in triplicate
samples by following the conversion of L-[
Assay conditions were as described under
``Experimental Procedures.'' Standard errors of the means are
indicated. Student's t test was used to compare values
corresponding to each group (addition or omission) versus to
the control (none). p values were <0.01 versus control.
Assays were performed in triplicate samples as described under
``Experimental Procedures.'' p < 0.01 for 0.1 or
1.0 mML-glutamate or 0.1 mM NMDA versus the control; p < 0.05 for glutamate plus MK-801 (or
ketamine or AP5 or glycine) versus control; p <
0.05 for NMDA plus EGTA (or MK-801 or ketamine or AP-5) versus control.
Assays were
performed as in Table III, except that 1 mML-arginine was used instead of the labeled amino acid,
and NO generation was measured by accumulation of
NO
Assays were performed in triplicates on duplicate
samples as indicated under ``Experimental Procedures.'' p < 0.05 for 0.1 mM arginine versus control; p < 0.01 for 1.0 mM arginine or nitroprusside or
NMDA or glutamate versus control.
Conditions were described under
``Experimental Procedures.'' p < 0.01 for glycine
or serine (with or without glutamate) versus control.
We acknowledge Dr. Alberto R. Kornblihtt for helpful
criticisms.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, calmodulin, tetrahydrobiopterin, and FAD, and
inhibited by N
-methyl-L-arginine. L-Glutamate and N-methyl-D-aspartate
stimulated in vivo conversion of L-arginine to
citrulline by epimastigote cells. These stimulations could be blocked
by EGTA, MK-801, and ketamine and enhanced by glycine. A sodium
nitroprusside-activated guanylyl cyclase activity was detected in
cell-free, soluble preparations of T. cruzi epimastigotes. L-Glutamate, N-methyl-D-aspartate, and
sodium nitroprusside increased epimastigote cyclic GMP levels. MK-801
bound specifically to T. cruzi epimastigote cells. This
binding was competed by ketamine and enhanced by glycine or L-serine. Evidence thus indicates that in T. cruzi epimastigotes, L-glutamate controls cyclic GMP levels
through a pathway mediated by nitric oxide.
(
)also known as
endothelium-derived relaxing factor(1, 2) . Within the
neural or endothelial cells where it originates or in neighboring
cells, NO activates heme-containing soluble guanylyl
cyclase(3, 4) , thereby acting either as an
intracellular or an intercellular signaling molecule. Consequently, NO
formation is associated with an increase in cyclic GMP
levels(5) .
(6, 7, 8, 9) . NO
synthases of the second group are induced at the transcriptional level
by bacterial toxins and some cytokines and are found in macrophages,
vascular smooth muscle cells, fibroblasts, and
hepatocytes(10, 11, 12) .
(13, 14) .
-stimulated NO synthase, a nitroprusside-activated
guanylyl cyclase, as well as NMDA receptors in epimastigote forms of
the parasite is demonstrated. In addition, evidence indicating that L-arginine and NMDA increase NO production and cyclic GMP
levels in epimastigote cells is also presented.
Materials
L-[2,3-H]Arginine
(53 Ci/mmol),
H-labeled
(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine
hydrogen maleate (MK-801, 20 mCi/nmol),
H-labeled
(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP,
40 mCi/nmol), [
-
P]GTP,
H-labeled cyclic GMP, and a cyclic GMP
I
radioimmunoassay kit were obtained from DuPont NEN. Amino acid analogs
were purchased from Research Biochemicals Inc. Components of the T.
cruzi growth medium were obtained from Difco, and AG50WX-8 resin
was obtained from Bio-Rad. Other reagents were purchased from Sigma.
Cell Culture and Homogenates
T. cruzi epimastigote forms (Tulahuen 2 strain) were cultured 7 days at 28
°C in a medium containing (per liter) Bacto Liver (35 g), tryptose
(10 g), yeast extract (3 g), glucose (5 g), NaHPO
(8 g), NaCl (4 g), KCl (0.4 g), and hemine (20 mg)(15) .
The pH was adjusted to 7.8. All the components of this medium were
autoclaved 15 min at 118 °C. Standing cultures were carried out 7
days at 28 °C up to the late-exponential phase in 1-liter
Erlenmeyer flasks containing 100 ml of medium.
g, washed three times with
0.25 M sucrose containing 5 mM KCl, and homogenized
in the same solution (10 ml g
of wet cells) with a
Sorvall Ribi press operated at 34.5 megapascal (5000
lb/in
) under a N
atmosphere.
Membrane Preparation
After cell
homogenization, the extract was centrifuged 15 min at 1000 g. The membrane pellet was resuspended in 0.25 M sucrose containing 5 mM KCl and layered onto a
discontinuous gradient containing 1.58, 1.90, and 2.20 M sucrose. After centrifugation in a Beckman SW-40 rotor for 60 min
at 90,000
g, membranes were recovered from the
interface of 1.58 and 1.90 M sucrose and stored at -70
°C.
NO Synthase Purification
After
homogenization, cell debris was discarded by centrifugation at 1000
g for 10 min. The supernatant fluid, adjusted to 0.5
mM phenylmethylsulfonyl fluoride, 25 units ml
aprotinin, 0.01% leupeptin (w:v), and 0.2 mg ml
soybean trypsin inhibitor, was further centrifuged 60 min at
105,000
g. The supernatant fluid, referred to as
``soluble crude extract,'' was immediately processed to avoid
proteolytic degradation.
10 cm) equilibrated with
50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA,
0.5 mM phenylmethylsulfonyl fluoride, and 25 units
ml
aprotinin (buffer A). The column was then washed
with 50 ml of buffer A and eluted with 100 ml of a linear gradient of
0-400 mM NaCl in buffer A. Fractions of 2 ml were
collected. The NO synthase activity peak (6 ml) was mixed with 1 ml of
2`5`-ADP-agarose slurry equilibrated with 10 mM Tris-HCl
buffer containing 1 mM EDTA and 5 mM mercaptoethanol
(buffer B). The mixture was shaken 12 h at 4 °C. The slurry was
then poured into a column assembled in a Pasteur pipette and washed
with 25 ml of buffer B containing 0.5 M NaCl followed by 10 ml
of buffer B. The column was then eluted with 3 ml of buffer B
supplemented with 10 mM NADPH.
NO Synthase Assay
Enzyme activity was
measured by following the conversion of L-[ H]arginine to [
H]citrulline
according to the procedure described by Bredt and Snyder(16) .
Incubation mixtures contained 50 mM Tris-HCl buffer, pH 7.5, 1
µML-[
H]arginine (0.2
µCi per assay), 0.1 mM CaCl
, 10 µM tetrahydrobiopterin, 1 µM FAD, 1 µM NADPH, 1 mM dithiotreitol, and 10 µg/ml bovine brain
calmodulin in a total volume of 0.1 ml. Incubations were performed 2
min at 25 °C and stopped by the addition of 2 ml of ice-cold 20
mM Hepes buffer, pH 5.5, containing 1 mM EDTA.
Samples were immediately applied to 2-ml columns of AG50WX-8 resin
(Na
form) and washed with 2 ml of water. Percolate
plus wash from each column (4 ml) was mixed with 12.5 ml of
Bray's scintillation mixture and counted for radioactivity.
Enzyme activity was proportional to incubation time for the first 2
min, as well as to the amount of soluble crude extract protein up to 1
µg per assay.
/MeOH/NH
OH/H
O (1:4:2:1;
vol:vol) according to Iyengar et al.(17) . Under these
conditions, no other radioactive product was detected.
In Vivo Conversion of L-[
T.
cruzi epimastigotes were resuspended in Krebs-Henseleit medium,
and aliquots (1 ml, about 10H]Arginine to
[
H]Citrulline and Production of NO
cells) were incubated 45 min
at 28 °C in the presence of 1 µML-[
H]arginine (1 µCi per assay).
After the addition of the indicated amino acid derivative, incubation
was continued for 15 min. Reactions were stopped by the addition of 0.1
ml of ice-cold 70% trichloroacetic acid (w:v). After three cycles of
freeze-thawing, mixtures were centrifuged 10 min at 1000
g, and the supernatant solutions were extracted 4 times with 4
ml of diethyl ether to eliminate trichloroacetic acid. Aliquots (0.2
ml) of the aqueous phases were then mixed with 2 ml of 20 mM Hepes-NaOH buffer, pH 6.0, and purified by passage through AG50WX8
columns as described above.
using the Griess reagent, as described by Bredt and
Snyder(16) . In this case, concentration of L-arginine
was 1 mM.
Determination of Cyclic GMP
Levels
Incubations of epimastigote cells were performed as
described above for 3 min in the presence of the indicated excitatory
amino acid. Reactions were then stopped and processed according to this
procedure. After extraction with diethyl ether, samples were subjected
to acetylation and assayed for cyclic GMP using a radioimmunoassay kit
(DuPont NEN) following the instructions of the manufacturer.
Guanylyl Cyclase Assay
Enzyme activity
was assayed following the conversion of
[-
P]GTP to
P-labeled cyclic
GMP. Incubation mixtures contained 50 mM Tris-HCl buffer, pH
7.5, 0.1 mM 3-isobutyl-1-methylxanthine, 1 mM cyclic
GMP, 5 mM MgCl
, 0.1 mM
[
-
P]GTP (specific activity, 200 cpm/pmol),
2 mM phosphocreatine, 0.2 mg of creatine kinase, and
appropriate volumes of the enzyme preparation. Assays were performed in
the presence or absence of 0.1 mM sodium nitroprusside in a
total volume of 0.1 ml. Incubations were performed 5 min at 30 °C
on triplicate samples. Reactions were stopped by the addition of 0.1 ml
of a solution containing 10 mM
H-labeled cyclic
GMP (1500 cpm/mmol) followed by boiling for 2 min. Cyclic GMP was
purified and counted for radioactivity as described by Birnbaumer et al. (18). Under these conditions, reactions were
proportional to the amount of enzyme protein and incubation time.
Receptor Binding Assays
Incubation
mixtures for the binding assay contained 10 mM Tris-HCl
buffer, pH 7.5, 10 nM to 100 µMH-labeled ligand (MK-801 or CPP), and approximately 50
µg of membrane protein or 10
epimastigote cells in a
final volume of 0.1 ml. Incubations were performed 90 min at room
temperature. The bound peptide was separated by filtration through
nitrocellulose disks (Schleicher & Schuell, BA-85). Nonspecific
binding was determined in the presence of 0.1 mM unlabeled
ligand. Binding constants were calculated according to Cuatrecasas and
Hollenberg(19) .
NO Synthase Activity in Cell-free
Preparations
A soluble NO synthase activity was purified
and characterized from cell-free extracts of T. cruzi epimastigotes. The purification protocol, which included ion
exchange chromatography on DEAE-cellulose and affinity chromatography
on ADP-agarose, was similar to the one employed for the purification of
the rat cerebellum enzyme (20). As a result of these steps, enzyme
activity was purified about 2000-fold (). NO synthase
specific activity in epimastigote soluble extracts was in the same
order of magnitude as that found for cerebral tissue(20) .
H]arginine to
[
H]citrulline by the purified T. cruzi enzyme required NADPH and was blocked by EGTA. Reaction could be
stimulated by calmodulin, tetrahydrobiopterin, flavin adenine
dinucleotide, and flavin adenine mononucleotide (). This
NO synthase activity was also blocked by N
-monomethyl-L-arginine;
half-maximal inhibition was observed at about 40 µM of
this amino acid derivative. Most of these properties are very similar
to those of the neural synthase(20) .
Conversion of L-[
I shows that in epimastigote
cells, conversion of L-[H]Arginine to
[
H]Citrulline and Production of NO by
Epimastigote Cells
H]arginine to
[
H]citrulline is stimulated by L-glutamate and by NMDA. The stimulation was effectively
blocked by EGTA and non-competitive NMDA antagonists such as MK-801 and
ketamine(21) . AP-5, which has been described as a competitive L-glutamate antagonist(22) , slightly decreased the
effects of this amino acid and NMDA. On the other hand, glycine, which
has been reported to be a potentiator of L-glutamate responses
at the level of the NMDA receptor(23) , enhanced the L-glutamate effect.
.
As shown in , 1 mML-arginine slightly
stimulated NO production. Under such conditions, L-glutamate
and NMDA efficiently increased NO
accumulation.
Modulation of Guanylyl Cyclase Activity and Cyclic
GMP Levels
Guanylyl cyclase activity was detected in
cell-free preparations from T. cruzi epimastigotes. Enzyme
specific activity in the soluble crude extract was about 1-2
pmol/min/mg protein. Specific activity increased approximately 5-fold
in the presence of 0.1 mM sodium nitroprusside, which acts as
a NO donor.
Receptor Binding
Studies
[H]MK-801 bound specifically
to T. cruzi epimastigote cells and membranes. Binding could be
displaced 95% by 0.1 mM unlabeled MK-801 or ketamide.
Displacement studies of the labeled ligand by the unlabeled compound
gave an estimated dissociation constant of 7
10
M and about 10
receptors per cell. As shown
in , [
H]MK-801 binding was strongly
enhanced by glycine and L-serine and only slightly by L-glutamate.
H]CPP to
epimastigote cells or membranes was also determined. The compound bound
poorly and nonspecifically, making it impossible to determine any
binding parameter.
and calmodulin dependence.
levels.
H]MK-801 binding capacity, strongly enhanced by
glycine and L-serine. It has been postulated that
[
H]MK-801 binds within the ion channel of the
NMDA receptor(25) .
-calmodulin-dependent NO synthase, and a
nitroprusside-stimulated guanylyl cyclase. The effectors of such a
pathway should be a cyclic GMP-dependent protein kinase and unknown
phosphate protein acceptors. The characteristics of these entities in T. cruzi and their cellular effects require further studies.
Table: Purification of NO synthase from T.
cruzi epimastigotes
H]arginine to
[
H]citrulline as described under
``Experimental Procedures.''
Table: Factors affecting T. cruzi NO synthase
activity
Table: Modulation by amino acid derivatives of the
conversion of L-[H]arginine to
[
H]citrulline by T. cruzi epimastigote
cells
Table: Effects of L-arginine and excitatory
amino acids on NO production by epimastigote cells
in the medium. p < 0.01
for arginine versus control or for arginine plus glutamate (or
NMDA) versus arginine.
Table: Influence of
amino acids and sodium nitroprusside on cyclic GMP levels in T. cruzi
epimastigotes
Table: Modulation of
[H]MK-801 binding in epimastigote
membranes by amino acids
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.