Modulation of Na(+)/K(+) ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis

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 (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2), we measured enzyme activity using increasing concentrations of H(2)O(2) and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H(2)O(2). To investigate the role of PKC in this signaling pathway, we observed the production of H(2)O(2) 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 H(2)O(2). Furthermore, we found that PKC activity is increased in the presence of H(2)O(2), and that in the presence of calphostin C, H(2)O(2) 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 H(2)O(2) opens new possibilities for understanding the signaling pathways of this parasite.