J. Biol. Chem., Vol. 266, Issue 36, 24351-24358, Dec, 1991

Calcium homeostasis in Trypanosoma brucei. Identification of a pH- sensitive non-mitochondrial calcium pool

L Ruben, A Hutchinson and J Moehlman
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275.

The objective of this study was to characterize mechanisms which maintain intracellular calcium homeostasis in bloodstream forms of Trypanosoma brucei. The identification of homeostatic pathways is required to understand signal transduction in these organisms. The fluorescent probes Fura-2, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein, and bisoxonal were used to measure intracellular calcium ([Ca2+]i), intracellular pH (pHi), and membrane potential, respectively. Homeostatic pathways maintained [Ca2+]i at 98 +/- 12 nM in the presence of 1.8 mM extracellular calcium despite a steady leak of calcium into the cell. The addition of 2.7 microM nigericin acidified the cytosol, depolarized the plasma membrane, and induced an approximate 3-fold increase in [Ca2+]i. The rise in [Ca2+]i could not be induced with valinomycin or gramicidin D under conditions where membrane depolarization occurred. By contrast, the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, elevated [Ca2+]i in a manner that was not additive with nigericin. Changes in pHi appeared to regulate [Ca2+]i since: 1) stepwise addition of K+ to nigericin- treated cells generated and incremental increase in pHi and concomitant decrease in [Ca]i; 2) addition of serum to nigericin-treated cells allowed simultaneous recovery of pHi and [Ca2+]i; and 3) addition of NH4Cl to untreated cells resulted in a biphasic change in pHi with corresponding biphasic change in [Ca2+]i. The rise in [Ca2+]i was derived from an intracellular pool which was not dependent on functional cytochrome oxidase, mitochondrial alternative oxidase, or the F0F1-ATPase. These data demonstrate that large quantities of calcium are reversibly stored in a non-mitochondrial pH-sensitive intracellular pool in T. brucei. We conclude that changes in pHi can serve to trigger calcium signals in these organisms.
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