Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ

Vercesi, Aníbal E.; Bernardes, Celene Fernandes; Hoffmann, Maria E.; Gadelha, Fernanda Ramos; Docampo, Roberto

doi:10.1016/s0021-9258(18)98703-x

Cited by 167 publications

(25 citation statements)

References 20 publications

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“…12,16,18 Most important, the mitochondrial respiration in skinned muscle fibers is functionally comparable to that of high-quality isolated mitochondria preparations. 12,21,22 Measurement of mitochondrial respiratory rates. Mitochondrial respiratory rates were determined with a Clark electrode (Yellow Springs Instruments, Yellow Springs, Ohio) in an oxygraph cell containing 15 to 20 muscle bundles in 3 mL of solution B at 34°C, with continuous stirring.…”

Section: Methodsmentioning

confidence: 99%

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

Pipinos

Sharov

Shepard

et al. 2003

Journal of Vascular Surgery

123

141

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Objective: Discrete morphologic, enzymatic and functional changes in skeletal muscle mitochondria have been demonstrated in patients with peripheral arterial disease (PAD). We examined mitochondrial respiration in the gastrocnemius muscle of nine patients (10 legs) with advanced PAD and in nine control patients (nine legs) without evidence of PAD. Methods: Mitochondrial respiratory rates were determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Muscle samples were obtained from the anteromedial aspect of the gastrocnemius muscle, at a level 10 cm distal to the tibial tuberosity. Mitochondria respiratory rate, calculated as nanoatoms of oxygen consumed per minute per milligram of noncollagen protein, were measured at baseline (V 0 ), after addition of substrates (malate and glutamate; (V SUB ), after addition of adenosine diphosphate (ADP) (V ADP ), and finally, after adenine nucleotide translocase inhibition with atractyloside (V AT ). The acceptor control ratio, a sensitive indicator of overall mitochondrial function, was calculated as the ratio of the respiratory rate after the addition of ADP to the respiratory rate after adenine nucleotide translocase inhibition with atractyloside (V ADP / V AT ). Results: Respiratory rate in muscle mitochondria from patients with PAD were not significantly different from control values at baseline (0.31 ؎ 0.06 vs 0.55 ؎ 0.12; P ‫؍‬ .09), but V sub was significantly lower in patients with PAD compared with control subjects (0.43 ؎ 0.07 vs 0.89 ؎ 0.20; P < .05), as was V ADP (0.69 ؎ 0.13 vs 1.24 ؎ 0.20; P < .05). Respiratory rates after atractyloside inhibition in patients with PAD were no different from those in control patients (0.47 ؎ 0.07 vs 0.45 ؎ P ‫؍‬ .08). Compared with control values, mitochondria from patients with PAD had a significantly lower acceptor control ratio (1.41 ؎ 0.10 vs 2.90 ؎ 0.20; P < .001). Conclusion: Mitochondrial respiratory activity is abnormal in lower extremity skeletal muscle in patients with PAD. When considered in concert with the ultrastructural and enzymatic abnormalities previously documented in mitochondria of chronically ischemic muscle, these data support the concept of defective mitochondrial function as a pathophysiologic component of PAD.

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Section: Methodsmentioning

confidence: 99%

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

Pipinos

Sharov

Shepard

et al. 2003

Journal of Vascular Surgery

123

141

View full text Add to dashboard Cite

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“…Oxygen consumption rate was determined as described [31], using a Clark-oxygen electrode (Hansatech, Norfolk, U.K.) thermostatized at 25 mC. Promastigotes were washed twice in Hanks' medium and resuspended in respiration medium (125 mM sucrose, 65 mM KCl, 1 mM MgCl # , 2n5 mM KH # PO % , 0n33 mM EGTA and 10 mM Tris\HCl pH 7n2) at a density of 2n5i10* parasites\ml and kept at 4 mC.…”

Section: Respiratory Activitymentioning

confidence: 99%

The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1–8)M(1–18), a synthetic cecropin A–melittin hybrid peptide

Díaz‐Achirica

Ubach

Guinea

et al. 1998

Biochemical Journal

102

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Reports on the lethal activity of animal antibiotic peptides have largely focused on bacterial rather than eukaryotic targets. In these, involvement of internal organelles as well as mechanisms different from those of prokaryotic cells have been described. CA(1-8)M(1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. Using Leishmania donovani promastigotes as a model system we have studied the mechanism of action of CA(1-8)M(1-18), its two parental peptides and two analogues. At micromolar concentration CA(1-8)M(1-18) induces a fast permeability to H+/OH-, collapse of membrane potential and morphological damage to the plasma membrane. Effects on other organelles are related to the loss of internal homeostasis of the parasite rather than to a direct effect of the peptide. Despite the fast kinetics of the process, the parasite is able to deactivate in part the effect of the peptide, as shown by the higher activity of the d-enantiomer of CA(1-8)M(1-18). Electrostatic interaction between the peptide and the promastigote membrane, the first event in the lethal sequence, is inhibited by polyanionic polysaccharides, including its own lipophosphoglycan. Thus, in common with bacteria, the action of CA(1-8)M(1-18) on Leishmania promastigotes has the same plasma membrane as target, but is unique in that different peptides show patterns of activity that resemble those observed on eukaryotic cells.

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“…Fluorescence changes were monitored using a Hitachi F‐4500, F7000 or F‐7100 spectrofluorometer with excitation of 495 nm and emission of 586 nm (slit 2.5 nm width). Calibration of ΔΨ was done as reported before 38 …”

Section: Methodsmentioning

confidence: 99%

“…The mitochondrial membrane potential in situ was determined spectrofluorometrically, using the known probe safranine O, as described previously. 32,37,38 Briefly, T cruzi epimastigotes (5 × 10 7 cells) were incubated at 28°C in reaction buffer (125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH buffer at pH 7.2, 1 mM MgCl 2 , 2.5 mM potassium phosphate [1.95 mL]) containing 5 mM succinate, 0.2% BSA, 50 µM EGTA, and 5 µM safranine O, and the reaction was started with digitonin (50 µM) to permeabilize the cells. ADP (250 µM), carboxyatractyloside (1.5 µM), and FCCP (4 µM) were added to the medium at different time points as indicated in each experiment.…”

Section: Mitochondrial Membrane Potentialmentioning

confidence: 99%

Trypanosoma cruzi Letm1 is involved in mitochondrial Ca ²⁺ transport, and is essential for replication, differentiation, and host cell invasion

et al. 2021

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Leucine zipper‐EF‐hand containing transmembrane protein 1 (Letm1) is a mitochondrial inner membrane protein involved in Ca2+ and K+ homeostasis in mammalian cells. Here, we demonstrate that the Letm1 orthologue of Trypanosoma cruzi, the etiologic agent of Chagas disease, is important for mitochondrial Ca2+ uptake and release. The results show that both mitochondrial Ca2+ influx and efflux are reduced in TcLetm1 knockdown (TcLetm1‐KD) cells and increased in TcLetm1 overexpressing cells, without alterations in the mitochondrial membrane potential. Remarkably, TcLetm1 knockdown or overexpression increases or does not affect mitochondrial Ca2+ levels in epimastigotes, respectively. TcLetm1‐KD epimastigotes have reduced growth, and both overexpression and knockdown of TcLetm1 cause a defect in metacyclogenesis. TcLetm1‐KD also affected mitochondrial bioenergetics. Invasion of host cells by TcLetm1‐KD trypomastigotes and their intracellular replication is greatly impaired. Taken together, our findings indicate that TcLetm1 is important for Ca2+ homeostasis and cell viability in T cruzi.

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Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ

Cited by 167 publications

References 20 publications

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1–8)M(1–18), a synthetic cecropin A–melittin hybrid peptide

Trypanosoma cruzi Letm1 is involved in mitochondrial Ca ²⁺ transport, and is essential for replication, differentiation, and host cell invasion

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Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ

Cited by 167 publications

References 20 publications

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

Abnormal mitochondrial respiration in skeletal muscle in patients with peripheral arterial disease

The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1–8)M(1–18), a synthetic cecropin A–melittin hybrid peptide

Trypanosoma cruzi Letm1 is involved in mitochondrial Ca 2+ transport, and is essential for replication, differentiation, and host cell invasion

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Trypanosoma cruzi Letm1 is involved in mitochondrial Ca ²⁺ transport, and is essential for replication, differentiation, and host cell invasion