Low Transmembrane Potential Research Articles

Amyloid-β-Dependent Inactivation of the Mitochondrial Electron Transport Chain at Low Transmembrane Potential: An Ameliorating Process in Hypoxia-Associated Neurodegenerative Disease?

Cerebral hypoperfusion-induced hypoxia, a condition that impairs oxygen utilization and thus ATP production by mitochondrial oxidative phosphorylation (oxphos), is thought to contribute to neural degeneration in Alzheimer's disease. However, hypoxia upregulates the generation of amyloid-β (Aβ), a group of peptides known to impair/inhibit the electron transport chain (ETC) of reactions that support oxphos in the inner mitochondrial membrane (IMM). This is a hypothesis paper that reconciles the hypoxia-induced upregulation of Aβ with Aβ's ETC-inhibiting action and, specifically, posits an oxphos-enhancing effect of this inhibition under conditions of newly developing or otherwise mild hypoxia. This effect is typically transient; that is, under conditions of prolonged or severe hypoxia, the oxphos-enhancing activity is overwhelmed by Aβ's well-known toxic actions on mitochondria and other cellular components. The hypothesis is motivated by evidence that the IMM transmembrane potential Ψm, an important determinant of ETC activity, exhibits heterogeneity, i.e., a range of values, among a given local population of mitochondria. It specifically proposes that during oxygen limitation, Aβ selectively inactivates ETC complexes in mitochondria that exhibit relatively low absolute values of Ψm, thereby suppressing oxygen binding and consumption by complex IV of the ETC in these mitochondria. This effect of Aβ on low-Ψm mitochondria is hypothesized to spare hypoxia-limited oxygen for oxphos-enabling utilization by the ETC of the remaining active, higher-Ψm local mitochondria, and thereby to increase overall ATP generated collectively by the local mitochondrial population, i.e., to ameliorate hypoxia-induced oxphos reduction. The protective action of Aβ hypothesized here may slow the early development of hypoxia-associated cellular deterioration/loss in Alzheimer's disease and perhaps other neurodegenerative diseases.

Features of leukocytes’ apoptosis and emoxypine succinate efficacy in case of combined trauma of the chest and both thighs in rats

Objective: This study aims to establish features of blood leukocytes’ apoptosis and substantiate the efficacy of emoxypine succinate applying in case of combined trauma of the chest and both thighs in rats.
 Materials and Methods: Analysis of cell samples to determine reactive oxygen species was evaluated by the flow laser cytometry method, using 2.7-dichlorodihydrofluorescein diacetate (Sigma Aldrich, Germany). The number of leukocytes with low mitochondrial transmembrane potential was evaluated by the flow laser cytometry method, using a kit of reagents «MitoScreen» («BD Pharmingen», USA). The number of apoptotic leukocytes was evaluated by the flow laser cytometry method, using a kit of reagents “ANNEXIN V FITC” (“Beckman Coulter”, USA). Emoxypine succinate to animals was injected intraperitoneally 1 time per day during 14 days from the first day of experiment in the dosage of 40 mg/kg.
 Results and Discussion: It was established the progressive, statistically significant increasing of Annexin V- positive cells percentage from the first day of the combined trauma of the chest and both thighs in rats with the highest values within 7-14 days of observation. On 28 day of experiment the reduction of apoptotic white blood cells percentage by 7.7% than the findings on 14 day was observed, but it remained 33.3% higher than control. The analysis of data in case of emoxypine succinate applying indicates that production of reactive oxygen species by leukocytes began to decline after 3 days of experiment and continued to decrease with maximum of action on 7 day. On 28 day of experiment the production of reactive oxygen species by leukocytes has decreased by 39.8 %; the percentage of leukocytes with low transmembrane potential has decreased by 34.6 % vs rats without medical treatment. At the same time the dynamics of FITC Annexin V- positive leukocytes changes in case of combined trauma of the chest and both thighs in rats and emoxypine succinate applying on 28 day of experiment has decreased by 16.7 % vs rats without medical treatment.
 Conclusion: One of important signaling pathways of apoptosis triggering in case of experimental combined trauma of the chest and both thighs is reactive oxygen species overproduction and disruption of the mitochondrial inner membrane due to the decreasing of transmembrane potential in 3-7 days of observation. Emoxypine succinate applying in post-traumatic period has a positive effect, characterized by decreasing of the production of reactive oxygen species, the percentage of leukocyte with low mitochondrial transmembrane potential and the percentage of FITC Annexin V- positive cells of leukocyte suspension. But the dynamics of FITC Annexin V- positive leukocytes changes leads us to believe that in the initiation and implementation of cell death in case of combined trauma apart mitochondrial, there are other mechanisms.
 Bangladesh Journal of Medical Science Vol.18(2) 2019 p.244-251

Effect of four different in vitro incubation temperatures on functional dynamics, process of capacitation and apoptosis in goat spermatozoa

The present study was intended to divulge the insights into in vitro thermal stress induced capacitation and apoptosis like changes in Barbari buck spermatozoa. Forty eight semen ejaculates were obtained from four adult fertile Barbari bucks, diluted with TALP and further divided into four aliquots. The aliquots were incubated for 2h at four different temperatures e.g., 34, 37, 38 and 40°C. Physical seminal attributes were evaluated by employing standard fluorescent and non fluorescent techniques. Capacitation and acrosome reaction status were evaluated by chlortetracycline assay (CTC) and immunolocalization of tyrosine phosphorylated proteins as indicator of capacitation, while, apoptosis like changes were evaluated by evaluating translocation of phosphatidyl serine and inner transmembrane mitochondrial potential. The percentage of spermatozoa showing capacitation and acrosome reaction was significantly higher (P<0.05) at 38 and 40°C than other incubation temperatures. Similarly, percentage of spermatozoa showing phosphorylation of tyrosine containing proteins was higher at 38 and 40°C. Significant (P<0.05) increase in spermatozoa apoptosis was also recorded in terms of low mitochondrial transmembrane potential and high exteriorization of phosphatidyl serine at 38 and 40°C. Moreover, significant (P<0.05) reduction in spermatozoa motility, viability and intact acrosome was recorded with rise in incubation temperature beyond 38°C. Therefore, it can be concluded that, high incubation temperature induces functional alterations in buck spermatozoa in terms of apoptosis like changes, capacitation through increased phosphorylation of tyrosine containing proteins and acrosome reaction in spermatozoa, and thereby may be responsible for decrease in the quality of spermatozoa.

Effect of Sulfated Polysaccharides from Brown Algae on Apoptosis of Human Peripheral Blood Lymphocytes.

We studied the influence of fucoidans from brown algae Fucus evanescens, Laminaria cichorioides and Laminaria japonica on apoptosis of human peripheral blood lymphocytes. It was demonstrated that fucoidans induced lymphocyte apoptosis, increased the proportion of cells with low mitochondrial transmembrane potential, and inhibit expression of Bcl-xL gene in blood lymphocytes. These findings suggest that lymphocyte apoptosis induced by the analyzed sulfated polysaccharides is mediated by mitochondrial pathway.

Mitochondrial signaling inSaccharomyces cerevisiaepseudohyphae formation induced by butanol

Yeasts growing limited for nitrogen source or treated with fusel alcohols form elongated cells--pseudohyphae. Absence of mitochondrial DNA or anaerobic conditions inhibits this process, but the precise role of mitochondria is not clear. We found that a significant percentage of pseudohyphal cells contained mitochondria with different levels of membrane potential within one cell. An uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), but not the ATP-synthase inhibitor oligomycin D, prevented pseudohyphal growth. Interestingly, repression of the MIH1 gene encoding phosphatase activator of the G2/M transition partially restores the ability of yeast to form pseudohyphal cells in the presence of FCCP or in the absence of mitochondrial DNA. At the same time, retrograde signaling (the one triggered by dysfunctional mitochondria) appeared to be a positive regulator of butanol-induced pseudohyphae formation: the deletion of any of the retrograde signaling genes (RTG1, RTG2, or RTG3) partially suppressed pseudohyphal growth. Together, our data suggest that two subpopulations of mitochondria are required for filamentous growth: one with high and another with low transmembrane potential. These mitochondria-activated signaling pathways appear to converge at Mih1p level.

Role of Reactive Oxygen Species and Bcl-2 Family Proteins in TNF-α-Induced Apoptosis of Lymphocytes

We studied the in vitro apoptosis-inducing effect of recombinant TNF-α (rTNF-α) on blood lymphocytes from healthy donors. rTNF-α-induced apoptosis was accompanied by an increase in the number of cells with low mitochondrial transmembrane potential, increased intracellular content of reactive oxygen species, reduced content of Bcl-2, Bcl-xL, and Bax proteins, and elevated Bad content. The molecular mechanisms of these changes are discussed.

The Transport Properties of the Cell Membrane Ion Channels in Electric Fields: Bethe Lattice Treatment

The interactive two-state model of cell membrane ion channels in an electric field is formulated on the Bethe lattice by means of the exact recursion relations. The probability of channel opening or maximum fractions of open potassium and sodium channels are obtained by solving a non-linear algebraic equation. Using known parameters for the conventional mean-field theory the model gives a good agreement with the experiment both at low and high trans-membrane potential values. For intermediate voltages, the numerical results imply that collective effects are introduced by trans-membrane voltage.

Apoptosis and biochemical biomarkers of stress in spiders from industrially polluted areas exposed to high temperature and dimethoate

The aim of this study was to evaluate the relations between apoptosis and the activity of antioxidant enzymes (superoxide dismutase; catalase) and quantitative changes in stress protein positive cells (Hsp70; metallothionein) in midgut glands of funnel web spiders Agelena labyrinthica (Agelenidae) and wolf spiders Pardosa lugubris (Lycosidae) exposed to high temperature and pesticide under laboratory conditions. The spiders were collected from two meadow ecosystems differently polluted with metals (Olkusz and Pilica, southern Poland). Under stress conditions, P. lugubris had fewer apoptotic cells in the midgut glands than A. labyrinthica. In P. lugubris from both sites, the observed increase in the percentage of metallothionein and Hsp70-positive cells, simultaneous with intensification of superoxide dismutase and catalase activity, suggests an anti-apoptotic function of those proteins in representatives of wandering spiders. In the midgut glands of A. labyrinthica, heat shock and dimethoate increased the number of Annexin V-positive cells as well as the amounts of mitochondria with low transmembrane potential (Δ Ψ m) versus the control. The changes in the percentage of MT- and Hsp70-positive cells in funnel web spiders were less than in wolf spiders. The absence of change in SOD and CAT activity in A. labyrinthica shows that the participation of those enzymes in antioxidant reactions is minimal in this species.

Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95 (APO-1/Fas).

We have previously shown that nitric oxide (NO) induces apoptosis in different human neoplastic lymphoid cells through caspase activation. Here we studied the NO-mediated apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastasis (MCF-7). NO donor glycerol trinitrate (GTN) induced apoptosis in both cell lines which was completely abrogated after pretreatment with the broad spectrum caspase inhibitor zVAD-fmk. NO triggered also a time-dependent activation of caspase-1, caspase-3, and caspase-6 in these cells. Moreover, NO caused a release of mitochondrial protein cytochrome c into the cytosol, an increase in the number of cells with low mitochondrial transmembrane potential and with high level of reactive oxygen species production. However, NO did not induce mRNA expression of CD95 (APO-1/Fas) ligand. FAS-associated phosphatase-1 (FAP-1) molecule was constitutively expressed at the mRNA level and did not show any changes upon NO treatment in both breast cancer cell lines. The expression of the pro-apoptotic protein Bax and of the anti-apoptotic protein Bcl-2 remained unchanged in MCF-7 and BT-20 cells upon GTN treatment. We suggest that the mechanism of NO-mediated activation of the caspase cascade and subsequent apoptosis in human breast cancer cells required mitochondrial damage (in particular, cytochrome c release, disruption of mitochondrial transmembrane potential and generation of reactive oxygen species) but not the activation of the CD95/CD95L pathway.

Carcinogenesis and the plasma membrane

Presented is a two-stage hypothesis of carcinogenesis based on: (1) plasma membrane defects that produce abnormal electron and proton efflux; and (2) electrical uncoupling of cells through loss of intercellular communication. These changes can be induced by a wide variety of stimuli including chemical carcinogens, oncoviruses, inherited and/or acquired genetic defects, and epigenetic abnormalities. The resulting loss of electron/proton homeostasis leads to decreased transmembrane potential, electrical microenvironment alterations, decreased extracellular pH, and increased intracellular pH. This produces a positive feedback loop to enhance and sustain the proton/electron efflux and loss of intercellular communication. Low transmembrane potential is functionally related to rapid cell cycling, changes in membrane structure, and malignancy. Intracellular alkalinization affects a variety of pH-sensitive systems including glycolysis, DNA synthesis, DNA transcription and DNA repair, and promotes genetic instability, accounting for the accumulation of genetic defects seen in malignancy. The abnormal microenvironment results in the selective survival and proliferation of malignant cells at the expense of contiguous normal cell populations.

Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

AbstractWe have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

Nitric Oxide–Induced Apoptosis in Human Leukemic Lines Requires Mitochondrial Lipid Degradation and Cytochrome C Release

We have previously shown that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through activation of caspases not only via CD95/CD95L interaction, but also independently of such death receptors. Here we investigated mitochondria-dependent mechanisms of NO-induced apoptosis in Jurkat leukemic cells. NO donor glycerol trinitrate (at the concentration, which induces apoptotic cell death) caused (1) a significant decrease in the concentration of cardiolipin, a major mitochondrial lipid; (2) a downregulation in respiratory chain complex activities; (3) a release of the mitochondrial protein cytochrome c into the cytosol; and (4) an activation of caspase-9 and caspase-3. These changes were accompanied by an increase in the number of cells with low mitochondrial transmembrane potential and with a high level of reactive oxygen species production. Higher resistance of the CD95-resistant Jurkat subclone (APO-R) cells to NO-mediated apoptosis correlated with the absence of cytochrome c release and with less alterations in other mitochondrial parameters. An inhibitor of lipid peroxidation, trolox, significantly suppressed NO-mediated apoptosis in APO-S Jurkat cells, whereas bongkrekic acid (BA), which blocks mitochondrial permeability transition, provided only a moderate antiapoptotic effect. Transfection of Jurkat cells with bcl-2 led to a complete block of apoptosis due to the prevention of changes in mitochondrial functions. We suggest that the mitochondrial damage (in particular, cardiolipin degradation and cytochrome c release) induced by NO in human leukemia cells plays a crucial role in the subsequent activation of caspase and apoptosis.

The effect of glycine in capacitation medium on IVF and further development rates in the cow

The present study was intended to divulge the insights into in vitro thermal stress induced capacitation and apoptosis like changes in Barbari buck spermatozoa. Forty eight semen ejaculates were obtained from four adult fertile Barbari bucks, diluted with TALP and further divided into four aliquots. The aliquots were incubated for 2 h at four different temperatures e.g., 34, 37, 38 and 40 °C. Physical seminal attributes were evaluated by employing standard fluorescent and non fluorescent techniques. Capacitation and acrosome reaction status were evaluated by chlortetracycline assay (CTC) and immunolocalization of tyrosine phosphorylated proteins as indicator of capacitation, while, apoptosis like changes were evaluated by evaluating translocation of phosphatidyl serine and inner transmembrane mitochondrial potential. The percentage of spermatozoa showing capacitation and acrosome reaction was significantly higher (P < 0.05) at 38 and 40 °C than other incubation temperatures. Similarly, percentage of spermatozoa showing phosphorylation of tyrosine containing proteins was higher at 38 and 40 °C. Significant (P < 0.05) increase in spermatozoa apoptosis was also recorded in terms of low mitochondrial transmembrane potential and high exteriorization of phosphatidyl serine at 38 and 40 °C. Moreover, significant (P < 0.05) reduction in spermatozoa motility, viability and intact acrosome was recorded with rise in incubation temperature beyond 38 °C. Therefore, it can be concluded that, high incubation temperature induces functional alterations in buck spermatozoa in terms of apoptosis like changes, capacitation through increased phosphorylation of tyrosine containing proteins and acrosome reaction in spermatozoa, and thereby may be responsible for decrease in the quality of spermatozoa.

Modified cell proliferation due to electrical currents.

In view of the evidence that electrical currents may enhance healing of chronic wounds and retard tumour growth it is suggested that these currents normalise cell proliferation. Additional support to this contention is given by two reports: one on healing of pressure sores in man and one on tumour growth retardation in mice. The effect of an ionic environment on the cell cycle is analysed. Finally a hypothesis attempting to explain the normalising effect of electrical currents on cell proliferation is proposed. It is known that non-dividing cells, e.g. mature neurons, have high transmembrane potential (TMP) whereas fast-dividing cells, e.g. cancerous cells, have low TMP. When a cell is exposed to an electrical field, one side of the cell becomes hyperpolarised while the opposite side is depolarised. Assuming a nonlinear relationship between TMP and the transmembrane ionic currents, it can be shown that in non-dividing cells their high TMP is lowered; whereas in cells with a high division rate, their low TMP is raised due to cell exposure to the external electrical field. These alterations in transmembrane potential could contribute to the normalisation of abnormal cell proliferation.

Qualitative analysis of intracellular characteristics of spontaneous neurons in the nucleus reticularis gigantocellularis of the cat

The intracellular characteristics of spontaneous neurons reticularis gigantocellularis (NRGC) of cats were evaluated. Apart from an irregular pattern of spontaneous discharge, we demonstrated that out of 895 successfully impaled NRGC neurons, 744 had a relatively low transmembrane potential of – 12 to – 40 mV. Many reticular neurons manifested spontaneous pre-potentials and post-spike depolarizations, from which action potentials were observed to arise. It is discussed that the spontaneous nature of NRGC neuronal discharge may be the result of continuous presynpatic bombardments because of the special anatomic and physiologic features of the reticular formation. Alternatively, the NRGC neurons may possess pacemaker-like membrane properties.

Determination of electrolytes--methodological problems.

To avoid errors in plasma potassium determination clenching of the fist should be avoided and heparinized blood should be used. The patient should be fasting and samples taken at the same time of the day. When evaluating intracellular potassium from plasma potassium acid based status should be considered. The clinical status should also be considered, since severely ill patients may have a low transmembrane potential leading to loss of cell potassium and increase in plasma potassium. Measurements of exchangeable sodium and potassium with radioactive tracers of whole body potassium (40K) are inprecise for determination of absolute cellular concentrations but more suitable for longitudinal studies. Determination of red cell electrolytes is of limited value, since these cells are not typical for the body cells. Electrolyte determination in leukocytes may give more accurate information. Muscle water and electrolytes can be determined in needle biopsy material. The concentration of intracellular electrolytes are expressed in relation to different bases of reference (FFS, GFFS, alkali-soluble protein, phosphorus, and intracellular water); the advantages and fallacies in using these references are discussed.