Abstract
Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.
Highlights
Leishmania spp. are a group of protozoa from the Trypanosomatidae family that causes a variety of diseases that can manifest in a cutaneous or visceral form depending on the species of Leishmania [1,2]
We found that protein kinase C (PKC) activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme
To verify if the presence of H2O2 could have a role in the proliferation of Leishmania amazonensis cells, we investigated the H2O2 generation profile during the growth curve of the parasite
Summary
Leishmania spp. are a group of protozoa from the Trypanosomatidae family that causes a variety of diseases that can manifest in a cutaneous or visceral form depending on the species of Leishmania [1,2]. As a bloodstream form, promastigotes of Leishmania are constantly exposed to free heme, a pro-oxidant molecule with signaling capacity that comes from the digestion of hemoglobin in the midgut of the insect vector [6,7,8,9]. Hydrogen peroxide (H2O2) is generated by the dismutation of the superoxide anion radical (O2_ˉ) and can cross cell membranes more . At low concentrations it may play an important role in cell signaling pathways through oxidation of specific target molecules [10,11,15]
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