Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1, nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2+, and (2) the effect of ionomycin, which cannot take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A1, nigericin, monensin, or NH4Cl. Inorganic PPi promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PPi collapsed by addition of the K+/H+ exchanger, nigericin, and eliminated by the PPi analogue, aminomethylenediphosphonate (AMDP). Both PPi hydrolysis and proton transport were dependent upon K+, and Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H+-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca2+]i in the nominal absence of extracellular Ca2+. Ionomycin was more effective in releasing Ca2+ from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H+-PPase and V-H+-ATPase in these organelles.

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