Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

Abstract

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca 2+ from isolated P. chabaudi parasites at micro-and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP 3 and therefore mobilize intracellular Ca 2+ in Plasmodium inside red blood cells.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

“…Thus higher concentrations may be necessary to elicit maximal amplitude and immediate responses at the level of inositol phosphate generation and Ca 2ϩ mobilization. Previous studies from our laboratory have shown that the effects of melatonin on parasite Ca 2ϩ release and synchronized progression through the cell cycle are blocked by the PLC inhibitor U73122 (13,14,40). Importantly, the activation of PLC by melatonin in P. falciparum is corroborated in this study without the use of pharmacological inhibitors and the potential nonspecific effects of these compounds.…”

“…As mentioned in the "Results," increases in inositol polyphosphates by melatonin concentrations below 100 M were not significant. Melatonin in the lower range is capable of exerting effects on life cycle progression when included in RBC malaria parasite cultures (13,40). However, these cell cycle progression effects of melatonin occur on a much slower timescale than the 20-min incubations in the present experiments, and the associated Ca 2ϩ increases are also slower and of lower amplitude (40).…”

“…These molecules were able to induce Ca 2ϩ release from cultured P. falciparum and P. chabaudi and importantly these responses were blocked by PLC inhibition and melatonin receptor antagonism (14). Similarly, the ability of melatonin and other tryptophan derivatives to synchronize P. falciparum cultures were also blocked by inhibition of PLC and melatonin receptors (13,14,40). Whereas, in the intraerythrocytic stages of P. berghei and P. yoelii, two rodent parasites that show asynchronous development (not linked to circadian rhythm) in vivo, melatonin does not modulate their cell cycle or elicit an elevation in intracellular Ca 2ϩ (41).…”

PfPKB, a Protein Kinase B-like Enzyme from Plasmodium falciparum

“…1 and 4C) suggest that the activation of PfPKB by CaM is dependent on calcium, it was important to investigate the mechanism via which parasitic calcium regulates PfPKB. Intracellular calcium levels of the parasite are tightly regulated in Plasmodium, and inhibitors of phospholipase C (PLC) (21), which block inositol 1,4,5-trisphosphate formation, prevent release of free calcium from intracellular parasite stores (22,23). To investigate the role of PLC in PfPKB activation, schizonts were incubated with either U73122, a specific inhibitor of PLC, or its less potent analogue U73322.…”

“…PLC inhibitor U73122 has been successively used in Plasmodium to block calcium release (22). Using this inhibitor we were able to demonstrate that PLC is the upstream regulator of PfPKB as it mediates calcium release crucial for PfPKB activation.…”