This study describes the possible role of Mg(2+)-dependent ecto-ATPase activity on the Trypanosoma cruzi-host cell interaction. Mg(2+)-dependent ecto-ATPase activity is observed on the cell body and flagellar membranes of the parasite and is about 20 times greater in trypomastigotes, as compared with epimastigotes. Suramin (a competitive antagonist of P2 receptors) and the impermeant agent 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS), both inhibitors of ecto-ATPases, strongly inhibited ATPase activity and the adhesion and internalization of both evolutive forms by mouse resident macrophages. Suramin inhibited the growth of epimastigotes, suggesting a direct participation of ecto-ATPase activity in this process. To overcome the presence of suramin in the culture medium during the time of growth, Mg(2+) ecto-ATPase activity was enhanced 4-fold, as compared with control parasites. The over-expression in enzyme activity was followed by a dramatic increase in the adhesion of epimastigotes to resident macrophages above the level observed for non-treated parasites.

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42 +/- 0.31 nmol Pi/h x 10(8) cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15 +/- 2.91 nmol Pi/h x 10(8) cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent Km for Mg-ATP2- was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive 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 P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.

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Suramin, a symmetrical polysulfonated naphthylamine derivative of urea, is a potent antagonist to some P 2 receptors (purine and pyrimidine receptors) and is able to inhibit a large number of cellular enzymes, such as ecto-ATPases, retrovirus reverse transcriptase, DNA polymerase, among others. It had initially been used in the treatment of sleeping sickness and cancer chemotherapy. Suramin treatment may cause several morphological changes, like axonal degeneration or axon atrophy, which may lead neurons to apoptotic cell death; alterations in cellular organization and disruption of plasma membrane of chick embryo neural retina and rat sertoli cells. Suramin also caused a dilatation of the rough endoplasmic reticulum of Trypanosoma rhodesiense.We have shown on previous studies that suramin inhibits T. cruzi epimastigotes and trypomastigotes Mg 2+ -ecto-ATPase activity. The incubation of both evolutive forms in the presence of the drug inhibits the adhesion and internalization of epimastigotes and trypomastigotes by resident macrophages. It was also observed that suramin inhibits the growth of epimastigotes, and may cause reservossomes swelling if epimastigotes are maintained in the presence of the drug for at least 1 hour. In this study we analyzed the effects of suramin on Y strain trypomastigotes. Parasites were cultivated in LLCMK 2 cells, in RPMI medium containing 2% of fetal calf serum and 500 µM of suramin. The presence of the drug did not affect the duration of T. cruzi intracellular cycle, although some of the LLCMK 2 presented a premature disruption, leaving some amastigote and epimastigote forms in the supernatant.Preliminary results show, by scanning electron microscopy, that trypomastigotes cultivated in the presence of suramin are shorter and broader when compared to control cells, presenting an average size of 11 x 1,4 nm, while control trypomastigotes present an average size of 14 x 1,1 nm. The video microscopy technique showed that some of the suramin treated trypomastigotes present slower movements when compared to control cells. We also observed that some of the obtained trypomastigotes present parts of the flagellum or the whole flagellum detached from cell body.