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 Ca2+, calmodulin, tetrahydrobiopterin, and FAD, and inhibited by N omega-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.

The trypanocidal action of green tea catechins against two different developmental stages of Trypanosoma cruzi is reported for the first time. This activity was assayed with the nonproliferative bloodstream trypomastigote and with the intracellular replicative amastigote parasite forms. An ethyl acetate fraction from Camellia sinensis green tea leaves, which contains most of the polyphenolic compounds and the maximal trypanocidal activity, was obtained by fractionation of the aqueous extract with organic solvents. The active compounds present in this extract were further purified by LH-20 column chromatography and were identified by highperformance liquid chromatography analysis with a photo diode array detector and gas chromatography coupled to mass spectroscopy. The following flavan-3-ols derivatives, known as catechins, were identified: catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate. The purified compounds lysed more than 50% of the parasites present in the blood of infected BALB/c mice at concentrations as low as 0.12 to 85 pM. The most active compounds were gallocatechin gallate and epigallocatechin gallate, with minimal bactericidal concentrations that inhibited 50% of isolates tested of 0.12 and 0.53 pM, respectively. The number of amastigotes in infected Vero cells decreased by 50% in the presence of each of these compounds at 100 nM. The effects of the catechins on the recombinant T. cruzi arginine kinase, a key enzyme in the energy metabolism of the parasite, were assayed. The activity of this enzyme was inhibited by about 50% by nanomolar concentrations of catechin gallate or gallocatechin gallate, whereas the other members of the group were less effective. On the basis of these results, we suggest that these compounds could be used to sterilize blood and, eventually, as therapeutic agents for Chagas' disease.Trypanosoma cruzi is the causative agent of Chagas' disease, which is a major endemic disease in South and Central America (29). Human hosts are infected either by the triatomide insect vector bite, by blood transfusion, or by congenital transmission. The chronic phase of the disease occurs several years after infection, with cardiac and gastrointestinal pathologies being the typical clinical manifestations (6).The main approach to the elimination of Chagas' disease in areas of endemicity is the control of transmission by the insect vector (28). On the other hand, crystal violet is the only effective chemoprotective agent for banked blood. However, the high level of toxicity of this drug has imposed severe restrictions on its use (7,19,21,22).Treatment of patients with Chagas' disease relies on two chemotherapeutic agents: benznidazole and nifurtimox. These two drugs have several limitations because they are effective but highly toxic during the acute phase of the disease and scarcely beneficial in the chronic phase (23, 25). The undesirable side effects associated with these classical trypanocidal...