J . Euk. MicrobioL. 44(2), 1997 pp. 155-156 8 1997 by the Society of Protozoologists

Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway

CLAUD10 PEREIRA,* CRISTINA PAVETO,,* JOAQUIN ESPINOSA,* GUILLERMO ALONSO,* MIRTHA M. FLAW=* and HECTOR N. TORRES*.'

*Institute de Investigaciones en Ingenieria GenPtica y Biologia Molecular, Consejo Nacional de Investigaciones Cientljkas y TPcnicas and Facultad de Ciencias Exactas y Naturales,

Universidad de Buenos Aires. Obligado 2490, I428 Buenos Aires, Argenrina

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N--methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.

Supplementary key words. Cyclic GMP, guanylyl cyclase, nitric oxide synthase, NMDAn-glutamate receptors.

ITRIC oxide (NO) is a signaling molecule with important N messenger functions in endothelium, macrophages and the nervous system [7, 131. In neural cells, nitric oxide, which is synthesized from L-arginine by a Ca2+-calmodulin-dependent synthase, activates a soluble guanylyl cyclase activity, leading to a rise in cyclic GMP levels [4, 171. Moreover, stimulation of L-glutamate/N-methyl-D-aspartate receptors in neural cells leads to the influx of CaZ+, which in turn activates nitric oxide synthase [2, 103. Recent studies from this laboratory provided the first demonstration that the nitric oxide signaling pathway is also present in the lower eukaryotic organism Trypanosoma cruzi, the ethiological agent of Chagas' disease [14j. A NO synthase was purified from epimastigote soluble extracts. The enzyme required NADPH, was activated by Ca*+, calmodulin, tetrahydrobiopterin and FAD, and inhibited by N-methyl-L-ar- ginine. Moreover, glutamate and N-methyl-D-aspartate (NMDA) stimulated in vivo conversion of L-arginine to citrul- line by epimastigote cells. These stimulations could be blocked by, MK-801 and ketamine and enhanced by glycine and serine. In addition, a sodium nitroprusside-activated guanylyl cyclase was also detected in cell-free, soluble epimastigote prepara- tions, and L-glutamate, NMDA and sodium nitroprusside in- creased cyclic GMP levels, in vivo [14]. Here we report that T. cruzi motility can be controlled by agonists, intermediaries and effectors of the NO signaling pathway.

MATERIALS AND METHODS Reagents. Amino acid analogs were purchased from Re-

search Biochemicals Incorporated (Natick, MA, USA) and components of the T. cruzi growth medium were obtained from Difco Laboratories (Detroit, MI, USA).

Cell cultures. Trypanosoma cruzi epimastigote forms of the Tulahuen 2 strain were cultured up to the late logaritmic phase (six days at 28°C) in a biphasic axenic medium [15].

Cell motility measurements. Cells were collected at 1,000 g for 10 min and washed three times with 0.25 M sucrose in 5 mM KCl. Cells were immediately resuspended in Krebs-Hen- seleit phosphate solution containing 11 mM glucose at a density of about 10' cells ml-I, and incubated 30 min at room temper- ature in the presence of the indicated compounds. Motile cells were counted in a Neubauer hemocytometer. Each value rep- resents the average of at least three readings, with one hundred cells scored in each reading. Cell viability in the cultures was evaluated by following occasional movement in a giving field during at least 5 min, as well as by the maintenance of the

To whom correspondence should be addressed. FAX: 54-1-786- 8578, Email: torres@proteus.dna.uba.ar

flagellar structure. Under the conditions selected most of 95 % of the cells were alive before and after treatment with the in- dicated compounds.

RESULTS AND DISCUSSION The effects of a number of compounds on T. cruzi epimas-

tigote motility are shown in Table 1. The proportion of motile cells can be enhanced by addition of L-arginine, the substrate of nitric oxide synthase [12]. This effect is blocked by Nu- methyl-L-arginine, a competitive inhibitor of the enzyme [l ll. N-methyl-D-aspartate (NMDA) and L-glutamate, two agonists of the NMDA subtype of L-glutamate ionotropic receptors [6, lo], also enhanced epimastigote motility. Dose-response curves for both agonists showed a response in the expected micromolar range, in accordance with the very low receptor affinity for these compounds [9]. This stimulation is blocked by the anti- convulsivant MK-801 ((+)-5-methyl- lO,ll-dihydro-5H-diben- zo[a,dJ cyclohept-5.10-imine) which is a potent noncompetitive NMDA antagonist [18], and is enhanced by L-serine and gly- cine. These last two amino acids have been reported to be po- tentiators of L-glutamate/NMDA responses at the level of the NMDA receptor [3].

In addition, sodium nitroprusside, a potent guanylyl cyclase stimulator [8] that can lead to the spontaneous formation of nitric oxide, also stimulated T. cruzi cell motility. Taken to- gether, these observations indicate that a stimulation of guanylyl cyclase activity mediated by NO may be responsible for the increase in epimastigote motility. This conclusion is strongly supported by the fact that 8-Br-cyclic GMP, a stable analog of cyclic GMP that specifically stimulates cyclic GMP-dependent protein kinase [l], also increases the motility of T. cruzi epi- mastigotes. This effect was not observed with other nucleotides such as 5' GMP, GDP or cyclic AMP.

As in the case of neural cells, T. cruzi NMDA receptors would be expected to be major entities controlling cytosolic Ca2+ levels [lo]. Moreover, the fact that nitric oxide generation from L-arginine is dependent on the Ca2+-calmodulin complex, might explain the inhibition of epimastigote motility by phen- otiazinic calmodulin blockers, as observed by this laboratory a decade ago 1161.

Data presented in this paper indicates that the same factors affecting the nitric oxide pathway in T. cruzi 1141, also influ- ence epimastigote motility. Data also suggests that the expected effector of this pathway might be a cyclic GMP-dependent pro- tein kinase acting on unknown phosphate protein acceptors.

Finally, it is important to note that a neural control mecha- nism such as the long-term potentiation involved in memory [5], has in Trypanosomatidae such an old evolutionary prece- dent. Both cases involve NMDA receptors, a Caz+/calmodulin-

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156 J. EUK. MICROBIOL., VOL. 44, NO. 2. MARCH-APRIL 1997

Table 1. Effects of different compounds on T. cruzi epimastigote motility.

Motile epi- Motility Assay (additions and concentrations) mastigotes (70) increase (%)

Control (none) 43.4 1- 1.6 0 0.1 mM L-arginine 72.0 2 l . l b 66.0 1 .O mM L-arginine 77.8 2 2.2b 79.3 0.1 mM L-arginine + 1 .O mM

0.001 mM L-glutamate 44.3 ? 3.0' -2.1 N--methyl-L-arginine 37.6 rt 1.0' - 13.3

0.01 mM L-glutamate 67.3 ? 3.2b 55.1 0.1 mM L-glutamate 72.0 2 3.0b 66.0 1.0 mM L-glutamate 76.8 -t 2Sb 77.0 0.1 mM L-glutamate + 1.0 mM

MK-801 43.6 t 1.W 1 .o 0.01 mM L-glutamate + 1.0 mM gly-

cine + 1.0 mM L-serine 81.2 rt 0.9" 87.0

0.01 mM N-methyl-D-aspartate 54.3 t 1.7b.' 25.0 0.1 mM N-methyl-D-aspartate 70.2 t 4.0b 61.7 1 .O mM N-methyl-D-aspartate 78.8 1- 2.2b 81.6 0.1 mM N-methyl-D-aspartate + 1.0

mM MK-801 44.2 2 0.3f 1 .o 0.01 mM N-methyl-D-aspartate + 1.0

mM glycine + 1 mM L-serine 62.0 1- 0.7b.g.f.' 42.9 1.0 mM glycine + 1.0 mM L-serine -4.1 1.0 mM MK-801 42.7 t 2.1" -1.7 1 .O mM Na nitropmsside 79.8 t 3.2b 83.9 1.0 mM 8-Br-cyclic GMP 69.9 1- 0.5b 61.0 1.0 mM db-cyclic AMP 44.5 2 3.4" 2.6

1.0 mM GDP 47.0 2 0.8" 8.4

0.001 mM N-methyl-D-aspartate 42.5 -t 1.7" -2.1

41.6 rt 3.2=

1.0 mM 5' GMP 42.3 t 3.0' -2.6

a Not significant in reference to control assay. b,c,d,e,f,g,h Significant in reference to control assay, or 0.1 mM L-ar-

ginine, or 0.1 mM L-glutamate, or 0.01 mM L-glutamate, or 0.1 mM N-methyl-D-aspartate, or 0.01 mM N-methyl-D-aspartate, or 1 .O mM glycine + 1.0 mM L-serine assays, respectively. Significances were P < 0.001, except whose significance was 0.001 < P < 0.01.

dependent NO synthase and a nitroprusside-stimulated guanylyl cyclase.

ACKNOWLEDGMENTS

This study was supported in part by the Tropical Disease Research Programme, World Health Organization (T80/18 1/14, 880 177), by the International Centre for Genetic Engineering and Biotechnology-Trieste, the Argentine-Brazilian Centre for Biotechnology, the Instituto Fatala Chaben and the Universidad de Buenos Aires. C. P., MMF, and HNT are Career Members of the Consejo Nacional de Investigaciones Cientificas y TCc- nicas (CONICET); J. E. and G. A. are Fellows of the CONI- CET, C. P. is a Fellow of the Universidad de Buenos Aires.

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Received 7-22-96, 10-29-96; accepted 11-25-96