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PubMed1988

Electrical excitability of artificial enzyme membranes. IV. Theoretical approach of the membrane potential of synthetic proteinic films.

This paper deals with the theoretical approach of the membrane potential of artificial proteinic film. Programming techniques using finite difference simulations for the steady state and transient solutions of the Nernst-Planck and Poisson equations were used and solved by the collocation and corrector methods. This approach allows one to calculate the membrane potential without any discontinuity between the Donnan and the diffusion potentials, the thickness of the boundary layers being automatically determined by the intrinsic properties of the solution and of the membrane. The theoretical results are compared with experimental potentials measured on proteinic artificial films.

Enzymes↗
PubMedDate not supplied

Effects of hyperthermia on the intracellular pH and membrane potential of Chinese hamster ovary cells.

The effects of hyperthermia (exposure to 41-47 degrees C) on the intracellular pH and membrane potential have been studied using Chinese hamster ovary HA-1 cells. Our goal was to determine whether intracellular pH changes or changes in membrane potential correlated with cell killing. The intracellular pH (pHi) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pHi reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for delta pH (pHi-pHo). The membrane potential difference (delta psi) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that delta psi falls to zero only upon prolonged exposure to temperatures above 43 degrees C. When the external pH was changed from normal values the drop in delta psi occurred more readily. Development of thermotolerance resulted in an increase in the time required to make delta psi change by half. The changes in delta psi were shown to be irreversible. When the proton electrochemical gradient (delta mu H+) was calculated using the measured values of delta psi and delta pH, the trends observed were the same as those seen for delta psi. The changes observed for pHi can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in delta psi and delta mu H+ may reflect the physical breakdown of the transmembrane H+ gradients, which may be the actual mechanical process of cell death. No correlation of cell survival with the measured parameters was observed.

Animals↗
PubMed1980

The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms.

Mitochondria from guinea-pig cerebral cortex incubated in the presence of Pi or acetate are unable to regulate the extramitochondrial free Ca2+ at a steady-state which is independent of the Ca2+ accumulated in the matrix. This is due to the superimposition on kinetically regulated Ca2+ cycling of a membrane-potential-dependent reversal of the Ca2+ uniporter. The latter efflux is a consequence of a low membrane potential, which correlates with a loss of adenine nucleotide loss from the matrix, enable the mitochondria to maintain a high membrane potential and allow the mitochondria to buffer the extramitochondrial free Ca2+ precisely when up to 200 nmol of Ca2+/mg of protein is accumulated in the matrix. The steady-state extramitochondrial free Ca2+ is maintained as low as 0.3 microM. The Na+-activated efflux pathway is functional in the presence of ATP and oligomycin and accounts precisely for the change in steady-state free Ca2+ induced by Na+ addition. The need to distinguish carefully between kinetic and membrane-potential-dependent efflux pathways is emphasized and the competence of brain mitochondria to regulate cytosolic free Ca2+ concentrations in vivo is discussed.

Adenosine Triphosphate↗
PubMed1992

Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia.

1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 M omega. The current-voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK < 0.001 were calculated using the Goldman-Hodgkin-Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na(+)-K+ pump by exposure to K(+)-free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3-5 mV. 3. In most neurones, depolarizing current pulses (0.5-1 s duration) elicited a single action potential of 85-100 mV, followed by an after-hyperpolarization of 200-500 ms. In 10-15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5-8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX-insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX-insensitive action potential was reduced, and after-hyperpolarization was replaced by after-depolarization upon substitution of internal K+ by Cs+. The after-hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization-activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady-state plateau which was maintained for > 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed2006

Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode.

Mitochondrial membrane potential (Deltapsi(m)) plays important roles in the normal function of cells and in pathobiochemical situations. The application of ion-selective electrodes for the measurement of Deltapsi(m) is important for studying normal biological reactions and pathways and mitochondrial diseases. We constructed and optimized a computerized device for real-time monitoring of the Deltapsi(m), which included modification of tetraphenylphosphonium (TPP(+))-selective membrane that improved reproducibility of the TPP(+)-selective electrode. Application of MATLAB software increased the sensitivity of the system. We tested our improved device for membrane potential measurements of isolated mitochondria (in absolute scale of millivolts). In addition, we assessed relative changes of Deltapsi(m) (as changes in TPP(+) concentration) of digitonin-permeabilized cells (hepatocytes, control transmitochondrial cybrids, HeLa G and BSC-40) after addition of substrates, inhibitors, and uncoupler of respiratory chain. Our system can be successfully used for studies of many aspects of the regulation of mitochondrial bioenergetics and as a diagnostic tool for mitochondrial oxidative phosphorylation disorders.

Animals↗
PubMed1999

Loss of mitochondrial membrane potential is dependent on the apoptotic program activated: prevention by R-2HMP.

Recent evidence suggests that the mitochondrial membrane potential begins to decrease well before the cells commit to apoptotic death. By using cultured cerebellar granule cells, two types of apoptosis can be induced, one by adding cytosine arabinoside (Ara-c; p53-dependent apoptosis) and one by lowering the K(+) concentrations of the medium (p53-independent apoptosis). Cultures show clear signs of increased apoptosis (chromatin condensation as visualized with bis-benzamide) after 12 hr which increases with time up to 24 hr. A fluorescent probe, chloromethyl-tetramethylrhodamine methyl ester (CMTMR), a lipophilic, potentiometric dye, which when introduced into the media accumulates within mitochondria in proportion to the mitochondrial membrane potential, was added at various time points after the induction of apoptosis. In Ara-c-induced apoptosis, there was a shift in the distribution of cell populations towards low-intensity CMTMR fluorescence, whereas in control and low-K(+) cultures, there was no such shift. This effect was observed as early as 6 hr after adding Ara-c. The antiapoptotic drug R-N-2-heptyl-N-methylpropargylamine hydrochloride (R-2HMP) reversed this loss of mitochondrial membrane potential in Ara-c-induced apoptosis; the effect was antagonized by the S-2HMP.

Alkynes↗
PubMed1979

Membrane potential, contractile activation and relaxation rates in voltage clamped short muscle fibres of the frog.

1. Voltage clamped short (approximately 1.5 mm) muscle fibres of the frog can develop maximum tension of 4.3 kg/cm2. 2. The time course of contractile responses to prolonged depolarization is markedly dependent on the fibre membrane potential. With sufficiently long pulses the responses present a plateau and a spontaneous relaxation phase. 3. At room temperature (20--22 degrees C), and at membrane potentials of -10 mV the plateau duration is about 2 sec and the spontaneous relaxation rate is 0.50 sec-1. At membrane potentials of -35 mV the plateau duration is 4.6 sec and the spontaneous relaxation rate is 0.28 sec-1. 4. When the fibres are depolarized at room temperature with relatively short pulses (less than 2 sec), the contractile responses are cut short at the end of the pulse, and the fibres relax at a rate (11 sec-1) which is independent on the pulse amplitude and duration. 5. The relaxation rate after a short pulse can be affected by membrane potential only in the region near the contractile threshold where further release of contractile activator is expected to occur. 6. The time course of contractile responses to prolonged depolarization may be shortened by conditioning depolarization. 7. The system responsible for the release of contractile activator may be tentatively described by three states, resting, activated, and inactivated, in analogy with the model proposed by Chandler, Rakowski & Schneider (1976) to describe the possible configurations of the potential dependent charge movement in muscle.

Animals↗
PubMed1988

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis.

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.

Adenosine Diphosphate↗
PubMed1984

Effect of membrane potential on the cellular uptake of 2-N-methyl-ellipticinium by L1210 cells.

Some quaternary ammonium derivatives of ellipticine are active antitumor drugs on both experimental and human tumors. Because of their positive charge, the cellular uptake of these molecules is expected to be influenced by the electric membrane potential. Experimental variations of the potential were produced by changing the external potassium concentration and the potassium permeability by the addition of valinomycin. Using the fluorescent lipophilic cationic dye 3,3-dihexyloxacarbocyanine iodide, the L1210 cell membrane potential was estimated at -35 mV by flow cytometric analysis, and the same technique was then used to study the effects of the membrane potential variations on 2-N-methyl-ellipticinium (NME) cellular uptake. Our results show that indeed NME uptake depends on the cell membrane potential, which might then influence its pharmacological properties.

Alkaloids↗
PubMed1992

Renal Na(+)-phosphate cotransport in X-linked Hyp mice responds appropriately to Na+ gradient, membrane potential, and pH.

To investigate the mechanism for the 50% decrease in Vmax of the high-affinity phosphate transport system in the renal brush-border membrane of X-linked Hyp mice, we compared the effects of external Na+ concentration, membrane potential, pH, phosphonoformic acid (PFA), and arsenate on Na(+)-Pi cotransport in brush-border membrane vesicles prepared from normal mice and Hyp littermates. The affinity of the Na(+)-Pi cotransport system for Na+ (apparent Km = 60 +/- 7 and 64 +/- 2 mM for normal and Hyp mice, respectively) and the Na(+)-Pi stoichiometry estimated from Hill plots (2.5 +/- 0.2 and 2.9 +/- 0.6 for normal and Hyp mice, respectively) were similar in brush-border membranes of both strains. Inside-negative membrane potential, generated by anions of different permeabilities, stimulated Na(+)-Pi cotransport and inside-positive membrane potential generated by valinomycin, and a K+ gradient (outside greater than inside) inhibited Na(+)-Pi cotransport to the same extent in brush-border membranes derived from normal mice and Hyp littermates. The pH dependence of Na(+)-Pi cotransport was similar in brush-border membrane vesicles of normal and Hyp mice. The ratio of Na(+)-Pi cotransport measured at pH 7.5 relative to that at pH 6.5 was 2.9 +/- 0.6 in normal mice and 2.9 +/- 0.7 in Hyp mice. PFA was a competitive inhibitor of Na(+)-Pi cotransport in brush-border membranes of both normal and Hyp mice. However, the apparent Ki for PFA was significantly lower in Hyp mice (0.31 +/- 0.01 and 0.19 +/- 0.02 mM in normal and Hyp mice, respectively, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment.

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.

Chemotaxis, Leukocyte↗
PubMed2001

Regional differences in anandamide- and methanandamide-induced membrane potential changes in rat mesenteric arteries.

The possibility that anandamide is an endothelium-derived hyperpolarizing factor was explored in the rat mesenteric vasculature by use of conventional microelectrode techniques. In the main mesenteric artery, anandamide and its more stable analog methanandamide hardly caused a measurable change in membrane potential of the smooth muscle cells, which promptly hyperpolarized to EDHF liberated by acetylcholine. Inhibition of endogenous anandamide breakdown by phenylmethylsulfonyl fluoride did not increase membrane responses to acetylcholine. The CB(1) receptor antagonist SR141716 did not significantly influence EDHF-mediated hyperpolarization except at extremely high concentrations. Smooth muscle cells of third to fourth order branches of the mesenteric artery, which have a more negative resting membrane potential and show smaller responses to acetylcholine, hyperpolarized by about 6 mV to both anandamide and methanandamide, whereas another CB(1) receptor agonist, WIN 55,212-2, had no effect. Mechanical endothelium removal or pre-exposure to SR141716A did not affect anandamide- and methanandamide-induced hyperpolarizations. However, in the presence of capsazepine, a selective vanilloid receptor antagonist, these membrane potential changes were reversed to a small depolarization, whereas EDHF-induced hyperpolarizations were not affected. Pretreating small vessels with capsaicin, causing desensitization of vanilloid receptors and/or depletion of sensory neurotransmitter, completely blocked methanandamide-induced hyperpolarizations. These findings show that anandamide cannot be EDHF. In smooth muscle cells of small arteries, anandamide-induced changes in membrane potential are mediated by vanilloid receptors on capsaicin-sensitive sensory nerves. The different membrane response to the cannabinoids between the main mesenteric artery and its daughter branches might be explained by the different density of perivascular innervation.

Animals↗
PubMed1997

Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase.

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a fluorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked fluorescent heterogeneity was observed in hepatocytes from old (20-28 months) but not young (3-5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 fluorescence pattern, with the largest population from old rats having significantly lower fluorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of -154 mV, in contrast to that of the three subpopulations from old rats of -70 mV (the largest population), -93 mV, and -154 mV. Production of oxidants was examined using 2',7'dichlorofluorescin, a dye that forms a fluorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an age-associated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.

Aging↗
PubMed1982

Calcium movement and membrane potential changes in the early phase of neutrophil activation by phorbol myristate acetate: a study with ion-selective electrodes.

To quantitate calcium movements and membrane potential changes in stimulated neutrophils, we have measured net fluxes of Ca2+ and of the lipophilic cation tetraphenyl phosphonium by a very sensitive ion-selective electrode system. Activation of neutrophils by 3 X 10(-8) M phorbol 12-myristate, 13-acetate induces a release of approximately 20% of total cell calcium, with an initial lag period of less than 10 s. The Ca2+ outflux is markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurs by activation of the ATP-driven Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induces a transiently increased exchange of medium 45Ca with cell calcium, which is measurable a few seconds after cell exposure to the stimulant and peaks at approximately 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) results in a marked depolarization, with a lag period of approximately 60 s. The rate and extent of depolarization are reduced by 40 and 65%, respectively, in a low Na+ medium but are not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarizes neutrophils without either modifying their resting oxidative metabolism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibits no effect on the oxidative metabolism of neutrophils, does not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.

Animals↗
PubMed1990

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes.

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.

Animals↗
PubMed1989

Effect of acetylcholine on membrane potential in toad dorsal root ganglion neurons and its underlying ionic basis.

Intracellular recordings were made to investigate the responses of membrane potential to acetylcholine (ACh) on neurons in isolated toad dorsal root ganglion (DRG). In the 73 neurons examined, 67 were of type A, and the remaining 6 of type C cell. The resting membrane potential of these two types of cells was -67.5 +/- 1.3 mV (means +/- SE). During the application of ACh (4 x 10(-4)-6 x 10(-4) mol/L), the changes in membrane potential were as follows: 1) hyperpolarization, with amplitude of 9.1 +/- 3.0 mV (means +/- SE; n = 23); 2) depolarization, with amplitude of 12.9 +/- 2.2 mV (means +/- SE; n = 20); 3) biphasic response, i.e., hyperpolarization with amplitude of 8.0 +/- 2.4 mV (means +/- SE) followed by depolarization with amplitude of 10.9 +/- 2.1 mV (means +/- SE) (n = 24); no effect (n = 6). The hyperpolarization induced by ACh was blocked by superfusion with atropine (1.3 x 10(-5) mol/L; n = 23), while ACh depolarization was blocked by the mixture of d-tubocurarine (1.4 x 10(-5) mol/L) and hexamethonium (1.4 x 10(-5) mol/L) (n = 18). When ACh caused hyperpolarization, the membrane conductance was increased by 13.8% and the reversal potential was about -96 mV (n = 3). TEA (20 mmol/L) superfusion enhanced ACh depolarization amplitude by 48.2 +/- 3.2% (means +/- SE; n = 6), and depressed ACh hyperpolarization amplitude by 79.4 +/- 4.3% (means +/- SE; n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed2006

Visualization of mitochondrial membrane potential and reactive oxygen species via double staining.

Quantitative and qualitative analysis of both generated reactive oxygen species (ROS) and mitochondrial membrane potential cannot be detected simultaneously. We here introduce a simple, new double staining method. We have successfully used this for several years utilizing cerium for ROS detection and JC-1 staining to assess the mitochondrial membrane potential. The resultant signals on laser confocal images can be localized in the same cells and can easily quantify them. We used a confocal microscope along with our new, combined staining method to both visualize mitochondrial membrane potential (DeltaPsim) and imaged ROS. These were quantified by JC-1 staining and by cerium ions with reflectance in a method modified in our laboratory. To test this double labeling technique we used PC 12 cells subjected to 1 h hypoxia and 24h re-oxygenization. We are able to produce a quantitative analysis of red/green signals of JC-1 that reflected the energy state of the cells. Cerium reflectance correlates with the amount of ROS release in the same cells. Significant differences have been calculated after hypoxia and re-oxygenation in both modality of the cell staining. The red/green ratio was 18.2+/-9.3 (n=30) in normoxic cells versus 1.65+/-0.9 (n=30) in the hypoxia/re-oxygenation group (p<0.05). In the same randomly selected cells the average cerium reflectance signal intensity was 2.5+/-1.2 (n=30) in the control group while 5.8+/-3.1 (n=30) in the hypoxia/re-oxygenation group (p<0.05). This assay, by characterizing hypoxic injury and re-oxygenization induced ROS production, offers a qualitative and quantitative method to detect the consequences of oxidative stress in experimental conditions and to detect different cell protective strategies.

Analysis of Variance↗
PubMedDate not supplied

Mitochondrial membrane potential selects hybridomas yielding high viability in fed-batch cultures.

Prior research (Follstad, B. D.; Wang, D. I. C.; Stephanopoulos, G. Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur. J. Biochem. 2000, 267, 6534-6540.) identified mitochondrial membrane potential (MMP) as a marker of hybridoma subpopulations resistant to apoptosis caused by a variety of apoptosis inducers. In this study, we investigated the viability of hybridoma cell cultures inoculated with cells of varying MMP in regular fed-batch operation. A hybridoma cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123 (Rh123). These subpopulations showed dramatic differences in their apoptotic death kinetics. Fed-batches inoculated with a high MMP subpopulation reached higher viable cell concentrations and viabilities that were maintained for prolonged periods of time relative to fed-batches inoculated with low MMP subpopulations. These results underline the heterogeneous nature of hybridoma cell cultures and suggest that mitochondrial physiology is a critical parameter determining culture performance.

Animals↗