Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria.

A.E Vercesi,S.N Moreno,C.F Bernardes,A.R Meinicke,E.C Fernandes,R Docampo

doi:10.1016/s0021-9258(18)52912-4

A.E Vercesi, S.N Moreno + Show 4 more

Open Access

https://doi.org/10.1016/s0021-9258(18)52912-4

Copy DOI

Abstract

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.

Highlights

Thapsigargin Causes Ca2+ Release and Collaposfethe Membrane Potential of Trypanosoma bruceiMitochondria in Situand of Isolated Rat Liver Mitochondria*
In addition,we have found no evidence for an gargin in Trypanosoma cruzi amastigotes and epimastigotes increase in intracellulaCra" by release of the ion fromusing fura-2-loaded cells (7), we re-examined the effect of intracellular stores bnyigericin, measuring changes in nigericin and thapsigargin on Ca2+homeostasis in T. brucei cytosolic Ca2+ by duawlavelength spectrofluorometry using fura-2-loaded and digitonin-permeabilized cells
Inordertobetter analyze the mitochondrial, Ref. 6) Ca2+uptake by digitonin-permeabilized effects of nigericin andthapsigarginonmitochondria, we studied the effect of these drugs onT. brucei procyclic trypomastigotes mitochondriai n situ

Summary

RESULTS

Phosphate, 1 mM MgC12,1.0 mM ATP, 40 pM arsenazo 111, 3.5 pM Ca*+, and trypomastigotes(0.7 mg of protein/ml) in a total volume. Digitonin (DIG, 20 p ~ )s,odium orthovanadate (VAN, 500 are able to increase intracelluClar2+in T. brucei bloodstream pM), thapsigargin (TG,[8] pM), nigericin (NIG,2.75 pM), and calcium trypomastigotes by releasing it from intracellular stores. Ther addition of thapsigargin did not change thisslow rate of fura-2-loaded T. brucei bloodstream trypomastigotes sus- Ca2+ releasecaused by vanadate, whereas addition of calcium pended inbuffer containing [Ca"] = 1mM had a fluorescence ionophore rapidly released the Ca2+taken up and theendogintensity corresponding to a cytosolic Ca2+ concentration in enous Ca2+. Taken concentrations of nigericin on the mitochondrialCa2+uptake together, these results indicate that theincrease in cytosolic (in the presenceof vanadate, Ref. 6) by digitonin-permeabil-. If nigericin was added instead of antimycin, a higher Ca2+ release than that observed with antimycin A was detected, indicating, as expected (2123), a clear effect on these mitochondria in situ.

Effect of Thapsigargin on Mitochondria

NIG or

DISCUSSION

Findings

LJ t RLM