scholarly journals Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ

1992 ◽  
Vol 284 (2) ◽  
pp. 463-467 ◽  
Author(s):  
A E Vercesi ◽  
R Docampo
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Initial Rate ◽  
Reaction Medium ◽  
Ruthenium Red ◽  
Permeabilized Cells ◽  
Safranine O

The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably by the endoplasmic reticulum, was completely inhibited by 500 microM-vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.

Effects of hydrogen peroxide and hypochlorite on membrane potential of mitochondria in situ in rat heart cells

1991 ◽  
Vol 69 (11) ◽  
pp. 1705-1712 ◽  
Author(s):  
Noburu Konno ◽  
K. J. Kako
Keyword(s):  
Hydrogen Peroxide ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Rat Heart ◽  
Mitochondrial Membrane ◽  
Isolated Rat Heart ◽  
Heart Mitochondria ◽  
High Concentrations ◽  
Isolated Rat

Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 ± 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium. Keywords: ATP, calcium, cardiomyocyte, cell defense, mitochondrial membrane potential, oxidant, triphenylmethylphosphonium.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

scholarly journals Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation

1984 ◽  
Vol 217 (2) ◽  
pp. 453-459 ◽  
Author(s):  
M D Brand ◽  
S M Felber
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Concanavalin A ◽  
Mitochondrial Membrane ◽  
Ph Gradient ◽  
Electrochemical Gradient ◽  
Inner Membrane ◽  
Mitogenic Stimulation ◽  
Ion Movements

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.

scholarly journals Perifosine Mechanisms of Action in Leishmania Species

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Atteneri López-Arencibia ◽  
Carmen Martín-Navarro ◽  
Ines Sifaoui ◽  
María Reyes-Batlle ◽  
Carolina Wagner ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Leishmania Species ◽  
Mechanisms Of Action ◽  
Content Type ◽  
Phosphatidylserine Externalization ◽  
Potential Use

ABSTRACT Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

scholarly journals The effect of α-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ

1982 ◽  
Vol 206 (3) ◽  
pp. 619-626 ◽  
Author(s):  
R J Davis ◽  
B R Martin
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Adrenergic Receptor ◽  
Mitochondrial Membrane ◽  
Fat Cell ◽  
Alpha Adrenergic Receptor ◽  
Low Concentrations ◽  
Adrenergic Antagonist ◽  
Potency Order

1. The accumulation of [3H]methyltriphenylphosphonium by isolated fat-cells was used to estimate the membrane potential of mitochondria in situ. 2. An alpha-adrenergic receptor-mediated decrease in the apparent accumulation of [3H]methyltriphenylphosphonium was observed. Methoxamine, clonidine and low concentrations of phenylephrine decreased the calculated mitochrondrial membrane potential without significantly raising cyclic AMP levels, adenylate cyclase activity or stimulating lipolysis. The agonist potency order was phenylephrine greater than methoxamine greater than clonidine. 3. The decrease in the calculated mitochondrial membrane potential caused by phenylephrine, clonidine and methoxamine was blocked by the alpha-adrenergic antagonist prazosin but not by yohimbine nor by the beta-antagonist propranolol. This suggests that the effect on the calculated mitochondrial membrane potential may be mediated by alpha 1-like receptors.

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