Features of mitochondrial energetics in living unicellular eukaryote Tetrahymena pyriformis. A model for study of mammalian intracellular adaptation

Abstract

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as Delta psi (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP(4-) and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low Delta psi value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the Delta psi-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.

Living unicellular eukaryote Tetrahymena pyriformis as a model for study

“…Thus the most part of the mitochondrial ΔP should be stored in the form of ΔpH. Additional evidence consistent with our assumption of low Δψ in TP mitochondria was obtained in the experiments with a Δψ-sensitive probe Mito Tracker Red [4,26]. In contrast to the experiments with other eukaryotic cells, in TP, we did not observe mitochondria with bright fluorescence.…”

“…The difference in uncoupling activity between FCCP and DNP (or oleic acid) remained nearly the same in the presence of 20-30 mM pyruvate (for details, see [4]). Strong stimulation of the respiration rate by FCCP attested to high ΔP in TP mitochondria.…”

“…Earlier we suggested that the decrease of Δψ during hibernation is due to a partial conversion of ΔP from Δψ to ΔpH form, that such conversion prevents the dangerous exhaustion of the intramitochondrial ATP pool [4].…”

“…Mito Tracker Red, in contrast with another Δψ-sensitive probe rhodamine 123, did not usually clear out mitochondria during fixation. The bright fluorescence of Mito Tracker Red is clearly discerned in mitochondria-like organelles inside the fixed cells [4,26], where this Δψ-sensitive probe is concentrated within the phospholipid area.…”

“…Previously we suggested that the "animal-like" unicellular eukaryotes Tetrahymena pyriformis when sampled in the stationary phase of growth and washed with a substrate-free medium (TP) may in many aspects model liver cells under the conditions of reduced oxidative metabolism [4].…”