title: Extracellular ATP triggers proteolysis and cytosolic Ca²⁺ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites.
abstract: BACKGROUND
  Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.
  
  
  METHODS
  Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca²⁺ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.
  
  
  RESULTS
  The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca²⁺-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).
  
  
  CONCLUSIONS
  The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.
authors.affiliation: Department of Physiology, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Butantan, 05508-900 São Paulo, SP Brazil.
authors.forename: Laura Nogueira
authors.initials: LN
authors.lastname: Cruz
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.forename: Alexandre
authors.initials: A
authors.lastname: Budu
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Holder
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
authors.forename: Célia Rs
authors.initials: CR
authors.lastname: Garcia
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010962:Plasmodium berghei / Q000187:drug effects* / Q000378:metabolism*; D010967:Plasmodium yoelii / Q000187:drug effects* / Q000378:metabolism*; D059748:Proteolysis; D015398:Signal Transduction; D013194:Staining and Labeling; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D010455:Peptides; D014966:Xanthenes; D000255:Adenosine Triphosphate; D002118:Calcium