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

Abstract

IP 3 -dependent Ca2؉ signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca 2؉ upon addition of exogenous IP 3 . In the present study, we investigated whether the IP 3 signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. Malaria, caused by the obligate Plasmodium parasite, infects over 300 million people annually and resistance to current antimalarial drugs is an increasing problem (1-5). The intraerythrocytic phase of Plasmodium falciparum, the most lethal human malaria parasite, is the primary cause of malaria morbidity and mortality. Therefore, arrest of the red blood cell (RBC) 4 stage of Plasmodium life cycle is a clear pharmaceutical target. The RBC cycle of P. falciparum occurs over a period of 48 h (the life cycles of other Plasmodium species are also multiples of 24 h) and consists of three stages of parasite development known as ring, trophozoite, and schizont. Proliferation occurs by lysis of the RBC to release merozoites, which are the product of the end of shizogony. This is followed by rapid reinvasion of uninfected RBCs to complete the cycle (6 -9). The ability to overcome host defenses relies upon the synchrony of merozoite release into the blood stream, usually at a specific time of day (10, 11). Therefore, key to P. falciparum survival is synchronous maturation within the RBC. Clear evidence supports a role of host circadian rhythm in this process, mediated by melatonin and/or related host hormones (12-15).Parasites like most eukaryotes, utilize second messenger signaling cascades involving Ca 2ϩ and cAMP to coordinate cell function (6, 14, 16 -20). The Ca 2ϩ signaling toolkit in vertebrates is now well characterized (21, 22) and genetic (18,23,24) and pharmacological studies (14, 25) are increasing our knowledge of the signaling proteins that are evolutionarily conserved from Apicomplexa (the Plasmodium phylum). To date, key components of the classical Ca 2ϩ release cascade have been described in Apicomplexeans; including sequences of four putative heptahelical receptors (26), G-proteins, implied by the sensitivity of gametogenesis to cholera and pertusis toxins (27) and sequences of PLC␦-like isoenzymes (23, 28). Furthermore, Ca 2ϩ pumps such as SERCA and a plethora of Ca 2ϩ -regulated proteins have been identified (18, 29 -33). A clear indication of the importance of Ca 2ϩ homeostasis and Ca 2ϩ regulated signaling events in these organisms. However, a canonical IP 3 receptor transcript has yet to be identified in the genome of any Apicomplexean. Nevertheless, pharmacological data clearly demonstrate P. falciparum and the rodent malaria parasite P. chabaudi maintain intracellular Ca 2ϩ stores (14,16,34) and IP 3 -dependent Ca 2ϩ release has been demonstrated in isolated, permeabilized P. chabaudi (35). Importantly, evid...

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Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

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