Ca2+ storage in Trypanosoma brucei: the influence of cytoplasmic pH and importance of vacuolar acidity

• Published in: Biochemical journal Vol. 310; no. 3; pp. 789 - 794
• Main Authors: Scott, D.A, Moreno, S.N.J, Docampo, R
• Format: Journal Article
• Language: English
• Published: England 15.09.1995
• Subjects: Ammonium Chloride - pharmacology; Animals; Anti-Bacterial Agents - pharmacology; calcium; Calcium - metabolism; cytoplasm; Cytosol - drug effects; Cytosol - metabolism; Enzyme Inhibitors - pharmacology; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; inorganic ions; Ionomycin - pharmacology; Ionophores - pharmacology; Kinetics; Macrolides; Nigericin - pharmacology; Propionates - pharmacology; Proton-Translocating ATPases - antagonists & inhibitors; Trypanosoma brucei; Trypanosoma brucei brucei - metabolism; trypomastigotes; vacuoles; Vacuoles - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3100789

The hypothesis that changes in cytosolic pH effect the release from intracellular compartments of stored calcium in Trypanosoma brucei was addressed by the use of procyclic and bloodstream trypomastigotes of T. brucei loaded with the fluorescent reagents 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) to measure intracellular pH (pH(i)), or fura 2 to measure intracellular free calcium ([Ca(2+)]i). Experiments were performed in EGTA-containing buffers, so increases in [Ca(2+)](i) reflected release of stored calcium rather than Ca(2+) entry. Nigericin reduced pH(i) and increased [Ca(2+)](i) in loaded cells, whilst propionate reduced pH(i), but did not affect [Ca(2+)](i), and NH(4)Cl increased both variables, so there appears to be no correlation between pH, and [Ca(2+)](i). Treatment of the cells with the calcium ionophore ionomycin under similar conditions (nominal absence of extracellular Ca(2+)) resulted in an increase [Ca(2+)](i) which was greatly increased by addition of either NH(4)Cl, nigericin or the vacuolar H+-ATPase inhibitor bafilomycin A(1). Similar results were obtained when the order of additions was reversed or when digitonin-permeabilized cells were used with the Ca(2+) indicator arsenazo III. The results suggest that more Ca(2+) is stored in this acidic compartment in procyclic than bloodstream forms. Taking into account the relative importance of the ionomycin-releasable and the ionomycin-plus-NH(4)Cl-releasable Ca(2+) pools, it is apparent that a significant amount of the Ca(2+) stored in T. brucei trypomastigotes is present in the acidic compartment thus identified.