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Inhibition of Ca2+ release from Trypanosoma brucei acidocalcisomes by 3,5-dibutyl-4-hydroxytoluene: role of the Na+/H+ exchanger.

ABSTRACT: Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+. They possess a vacuolar-type H+-ATPase for H+ uptake, a Ca2+/H+ countertransporting ATPase for Ca2+ uptake and a Ca2+/nH+ antiporter for Ca2+ release. Trypanosoma brucei procyclic trypomastigotes acidocalcisomes possess, in addition, an Na+/H+ antiporter that may participate in Ca2+ release from these organelles. In this work we show that the hydrophobic antioxidant 3,5-dibutyl-4-hydroxy toluene (BHT), at concentrations in the range 1-20 microM, inhibits Na+-induced Ca2+ release from the acidocalcisomes of digitonin-permeabilized procyclic trypomastigotes in a concentration-dependent manner. This effect supports the notion that Ca2+ release from this compartment is regulated by the activity of the Na+/H+ antiporter. In the presence of BHT, Ca2+ release could still be obtained by nigericin-mediated alkalinization of the acidocalcisomes, clearly demonstrating that the action of BHT is not at the level of the Ca2+/nH+ antiporter but on that of the Na+/H+ antiporter. In the same range of concentrations and depending on the preincubation time, BHT had an stimulatory or an inhibitory effect on the vacuolar H+-ATPase present in T. brucei acidocalcisomes. Since these effects of BHT were obtained at concentrations which are commonly used for its antioxidant properties, these results indicate that care should be exercised when attributing effects of BHT to only these properties.

SUBMITTER: Vercesi AE

PROVIDER: S-EPMC1218944 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Sodium-proton exchange stimulates Ca2+ release from acidocalcisomes of Trypanosoma brucei.

Project description:Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+ [Vercesi, Moreno and Docampo (1994) Biochem. J. 304, 227-233; Scott, Moreno and Docampo (1995) Biochem. J. 310, 789-794; Docampo, Scott, Vercesi and Moreno (1995) Biochem. J. 310, 1005-1012]. The data presented here indicate that Na+ stimulates Ca2+ release from the acidocalcisomes of digitonin-permeabilized Trypanosoma brucei procyclic trypomastigotes in a dose-dependent fashion, this effect being enhanced by increasing pH of the medium from 7.0 to 7.8. The hypothesis that this Na+ effect was mediated by alkalinization of the acidocalcisomes via a Na+/H+ antiporter was supported by experiments showing that Na+ promotes release of Acridine Orange previously accumulated in these vacuoles. This putative antiporter did not transport Li+ and was not sensitive to the amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride. Addition of the Na+/H+ ionophore monensin to intact cells loaded with fura 2, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, was followed by an increase in cytosolic Ca2+ concentration ([Ca2+]i), which was more accentuated in the presence of extracellular Na+. An increase in intracellular pH (pHi) of BCECF-loaded cells was detected after addition of monensin in the presence of extracellular Na+, whereas a dramatic decrease in pHi was detected in its absence, thus indicating the presence of a significant amount of releasable protons in the acidic compartments. These results are consistent with the presence of a Na+/H+ antiporter in the acidocalcisomes that could be involved in the regulation of pH1 and [Ca2+]1 in these parasites.

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TbIRK is a signature sequence free potassium channel from Trypanosoma brucei locating to acidocalcisomes.

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