©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Nitric Oxide Transduction Pathway in Trypanosoma cruzi(*)

Cristina Paveto (1)(§), Claudio Pereira (1), Joaquin Espinosa (1), Andrea E. Montagna (1)(¶), Marisa Farber (1)(¶), Mónica Esteva (2), Mirtha M. Flawiá (1)(§), Héctor N. Torres (1)(§)(**)

From the (1)Instituto de Investigaciones en Ingeniera Genética y Biologa Molecular, CONICET, and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires 1428, Argentina and (2)Instituto Nacional de Diagnóstico e Investigación de la Enfermedad de Chagas ``Mario Fatala Chabén,'' Buenos Aires 1063, Argentina

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

In mammalian cells, L-arginine is metabolized to yield nitric oxide (NO),()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) .

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(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) .

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(13, 14) .

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-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.


EXPERIMENTAL PROCEDURES

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.

Cells were collected by centrifugation at 1000 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.

The soluble crude extract (25 ml) was loaded onto a DEAE-cellulose column (1 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.

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/MeOH/NHOH/HO (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-[H]Arginine to [H]Citrulline and Production of NO

T. cruzi epimastigotes were resuspended in Krebs-Henseleit medium, and aliquots (1 ml, about 10 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.

NO generation by epimastigote cultures was monitored by the formation of NO 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 mMH-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) .


RESULTS

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) .

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 [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-[H]Arginine to [H]Citrulline and Production of NO by Epimastigote Cells

I shows that in epimastigote cells, conversion of L-[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.

The effect of excitatory amino acids was also studied by monitoring the concentration of NO in the incubation medium as accumulation of NO. 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.

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.

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 10M 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.

The binding of [H]CPP to epimastigote cells or membranes was also determined. The compound bound poorly and nonspecifically, making it impossible to determine any binding parameter.


DISCUSSION

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 and calmodulin dependence.

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 levels.

A well known feature of neural NMDA receptors is also found in T. cruzi epimastigote membrane receptors. This is the [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) .

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-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

NO synthase activity was assayed in triplicate samples by following the conversion of L-[H]arginine to [H]citrulline as described under ``Experimental Procedures.''


  
Table: Factors affecting T. cruzi NO synthase activity

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.


  
Table: Modulation by amino acid derivatives of the conversion of L-[H]arginine to [H]citrulline by T. cruzi epimastigote cells

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.


  
Table: Effects of L-arginine and excitatory amino acids on NO production by epimastigote cells

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 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

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.


  
Table: Modulation of [H]MK-801 binding in epimastigote membranes by amino acids

Conditions were described under ``Experimental Procedures.'' p < 0.01 for glycine or serine (with or without glutamate) versus control.



FOOTNOTES

*
This study was supported in part by the TDR Programme, World Health Organization, T80/181/14, 880177, by the International Centre for Genetic Engineering and Biotechnology-Trieste, and by the Fundación Antorchas (Argentina). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Career members of the Consejo Nacional de Investigaciones Cientficas y Técnicas (CONICET).

Fellows of the CONICET.

**
To whom correspondence should be addressed: INGEBI, Obligado 2490, Buenos Aires 1428, Argentina. Tel.: 541-784-5516; Fax: 541-786-8578.

The abbreviations used are: NO, nitric oxide; NMDA, N-methyl-D-aspartate; MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate; CPP, (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid.


ACKNOWLEDGEMENTS

We acknowledge Dr. Alberto R. Kornblihtt for helpful criticisms.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.