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Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from L: -arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2', 5'-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (+/-6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.

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The present report explores a comparative analysis of nitric oxide (NO(*)) production by three different species of Leishmania (L. amazonensis, L. braziliensis and L. chagasi). Among these species, L. braziliensis produced the highest amount of NO(*), measured in the supernatants of promastigotes cultures as nitrite, a stable by-product derived from NO(*). We have previously described the expression of a constitutive nitric oxide synthase (cNOS) in L. amazonensis promastigotes and axenic amastigotes. Comparing those results with the present work, using immunofluorescence assay, it was shown that both L. braziliensis and L. chagasi also express a cNOS. Immunostaining experiments showed that promastigotes from early passages of these species in culture had a strong immunoreactivity against anti-cNOS and anti-endothelial cell NOS, in comparison with the same parasite cultured for long time, suggesting a correlation between the NO(*) production and the presence of metacyclic forms prominent in those newly isolated parasites. These data corroborate findings of a higher NO(*) production by those parasites, following the growth curve. The relationship between the two NO(*)-generating systems in the parasite and in their host cell warrants further investigation. The presence of cNOS raises the possibility of a similar type of cross-talk or down-regulation between the NO(*) signaling systems in host cells and the lower eukaryotic-like Leishmania sp.