Experimental and functional changes in transmembrane potential and zeta potential of single cultured cells

Experiments were performed on single cells to investigate the relations between the total bioelectrical potential difference (PD) across the cell membrane (so-called transmembrane potential) and the net negative surface charge of the cell (zeta potential). The experiments were carried out on FL-cells, leucocytes and ovarian tumour cells. The PD was measured electrophysiologically by means of intracellular glass microelectrodes; the surface charge or the zeta potential was determined using cell electrophoresis. Both measuring methods are critically discussed.Under different conditions (hypothermia, hyperthermia, mitotic blocking agent, cell cycle), the transmembrane potential and zeta potential showed changes in an identical direction and often the response of transmembrane potential was found to be quicker and more intensive than that of the zeta potential. In other experiments (e.g. changing the extracellular Cl^? ion concentration) the reactions of both potentials showed no coincidence. Depending on the type of functionally or experimentally borne changes on the cytoplasmatic membrane, either both potentials or only one of them may be altered.     

Effects of electroporation on optically recorded transmembrane potential responses to high-intensity electrical shocks

The outcome of defibrillation shocks is determined by the nonlinear transmembrane potential (DeltaVm) response induced by a strong external electrical field in cardiac cells. We investigated the contribution of electroporation to DeltaVm transients during high-intensity shocks using optical mapping. Rectangular and ramp stimuli (10-20 ms) of different polarities and intensities were applied to the rabbit heart epicardium during the plateau phase of the action potential (AP). DeltaVm were optically recorded under a custom 6-mm-diameter electrode using a voltage-sensitive dye. A gradual increase of cathodal and well as anodal stimulus strength was associated with 1) saturation and subsequent reduction of DeltaVm; 2) postshock diastolic resting potential (RP) elevation; and 3) postshock AP amplitude (APA) reduction. Weak stimuli induced a monotonic DeltaVm response and did not affect the RP level. Strong shocks produced a nonmonotonic DeltaVm response and caused RP elevation and a reduction of postshock APA. The maximum positive and maximum negative DeltaVm were recorded at 170 +/- 20 mA/cm2 for cathodal stimuli and at 240 +/- 30 mA/cm2 for anodal stimuli, respectively (means +/- SE, n = 8, P = 0.003). RP elevation reached 10% of APA at a stimulus strength of 320 +/- 40 mA/cm2 for both polarities. Strong ramp stimuli (20 ms, 600 mA/cm2) induced a nonmonotonic DeltaVm response, reaching the same largest positive and negative values as for rectangular shocks. The transition from monotonic to nonmonotonic morphology correlates with RP elevation and APA reduction, which is consistent with cell membrane electroporation. Strong shocks resulted in propidium iodide uptake, suggesting sarcolemma electroporation. In conclusion, electroporation is a likely explanation of the saturation and nonmonotonic nature of cellular responses reported for strong electric stimuli.     

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

To evaluate therelationship between osteoblast differentiation and bioenergetics,cultured primary osteoblasts from fetal rat calvaria were grown inmedium supplemented with ascorbate to induce differentiation. Beforeascorbate treatment, the rate of glucose consumption was 320 nmol · h¹ · 10⁶cells¹, respiration was 40 nmol · h¹ · 10⁶cells¹, and the ratio of lactate production to glucoseconsumption was ~2, indicating that glycolysis was the main energysource for immature osteoblasts. Ascorbate treatment for 14 days led toa fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with thatshown in immature cells. Confocal imaging of mitochondria stained witha transmembrane potential-sensitive vital dye showed that mature cellspossessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain thecellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

     

Effects of reduced PO2 on rapid-drive-induced hyperpolarization of diastolic transmembrane potential in feline cardiac Purkinje strands

Using standard microelectrode techniques, we evaluated effects of diminished oxygen tension on the magnitude and time course of frequency dependent changes in maximum diastolic transmembrane potential (MDP) and on alteration of action potential duration (APD) in feline Purkinje fibers. MDP was recorded continuously during a control period (cycle length (CL) = 1000 ms), during a 5-min period of rapid drive (CL = 400 ms) and following return to pacing CL = 1000 ms. Rapid drive resulted in hyperpolarization of MDP from control value; and after return to pacing CL = 1000 ms. MDP gradually depolarized, eventually attaining a steady state value within +/- 0.5 mV of the control value. The difference between hyperpolarized MDP value and final steady-state value was designated VH, and the decline of MDP towards steady-state value approximated an exponential function (time constant = tau VH). Exposure to reduced PO2 (75 +/- 2.1 mmHg vs. control 473 +/- 39.1 mmHg) (1 mmHg = 133.322 Pa) resulted in reduction in the magnitude of VH (6.2 +/- 3.43 mV vs. 7.8 +/- 2.73 mV, mean +/- SD, p less than 0.005) and shortening of APD within 0-24 min, while measurable prolongation of tau VH (75 +/- 18.5 vs. 54 +/- 9.0 s, p less than 0.005) began at 25-49 min following onset of reduced PO2. These observations suggest that rate-related changes of MDP in cardiac tissues are oxygen dependent, and they support previously reported analagous observations in nerve which suggested that frequency dependent potential changes may in part reflect alterations of electrogenic Na-K pump activity.     

Prostaglandin modulation of N-formylmethionylleucylphenylalanine-induced transmembrane potential changes in rat neutrophils

Prostaglandins of the E-series (PGEs) and PGI2 will inhibit formylmethionylleucylphenylalanine-(f-Met-Leu-Phe) induced lysosomal enzyme release and superoxide-anion (O2-) production by neutrophils. The inhibitory effects of PGEs and PGI2 on neutrophil functional responses have been correlated with their ability to increase intracellular cAMP. In this study we have examined the effects of PGEs and PGI2 on f-Met-Leu-Phe- and phorbol-myristate-acetate-induced rat neutrophil membrane potential changes using an optical probe of membrane potential 3,3-dipropylthiodicarbocyanine iodide. 15-(S)-15-methyl-PGE1 (15-methyl-PGE1), a stable analogue of PGE1 and PGI2 inhibited f-Met-Leu-Phe-induced transmembrane potential changes in a dose-dependent manner. This inhibition was correlated with the ability of these agents to increase intracellular cAMP levels and inhibit O2- production and degranulation. In contrast, 15-methyl-PGE1 and PGI2, did not inhibit phorbol-myristate-acetate-induced transmembrane potential changes and O2- production. These results suggest independent mechanisms of activation of neutrophils by phorbol myristate acetate and f-Met-Leu-Phe, and they also suggest that the inhibitory effects of prostaglandins and cAMP on f-Met-Leu-Phe-stimulated cells is at a step or steps prior to activation of those processes involved in effecting changes in transmembrane potential, which are common to both stimuli.     

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads.

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.     

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (Delta rho) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial Delta rho and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 mu g ml-1 for 40 min for Rhodamine, and 1 mu S mD for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. ‘Round-shaped’ osteoclasts had significantly higher mitochondrial Delta rho and volume in the apical regions than in the basolateral portions (p 0.00001). In contrast, mitochondrial Delta rho and volume in ‘irregular- shaped’ osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption. This revised version was published online in November 2006 with corrections to the Cover Date.     

Low-temperature induced transmembrane potential changes in the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that the liverwort Conocephalum conicum generates all-or-none action potentials (APs) in response to a sudden temperature drop. In plants with anion and potassium conductance blocked, dose-dependent voltage transients (VTs) were evoked by cold stimuli. These VTs did not propagate. When the external concentration of Ca(2+) was decreased or calcium channel inhibitors (La(3+), Gd(3+), verapamil, Mg(2+), Mn(2+)) were used, inhibition of VTs was observed. Amplitudes of both APs and VTs grew when Sr(2+) ions, known to release calcium from internal stores, were added to the medium. Neomycin, which suppresses phospholipase C and indirectly affects inositol triphosphate formation, caused substantial inhibition of both APs and VTs. It is concluded that a temperature drop elucidated membrane potential changes due to calcium influx both from external and internal stores.     

The morphological changes in cultured cells caused by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis is the causative agent for human whooping cough. It was found that Bordetella pertussis infection caused a change in shape from flat to round in L2 cells, which are derived from rat type 2 alveolar cells. This phenomenon was reproduced using the culture supernatant of B. pertussis, and bacterium-free adenylate cyclase toxin (CyaA) was identified as the factor responsible. A purified preparation of wild-type CyaA but not an enzyme-dead mutant caused the cell rounding. It was examined whether CyaA causes similar morphological changes in various cultured cell lines. L2, EBL, HEK293T, MC3T3-E1, NIH 3T3, and Vero cells were rounded by the toxin whereas Caco-2, Eph4, and MDCK cells were not, although all these cells showed a significant elevation of the intracellular cAMP level in response to CyaA treatment, which indicates that there is no quantitative correlation between the rounding phenotype and the intracellular cAMP level. CyaA has been believed to target various immunocompetent cells and support the establishment of the bacterial infection by subverting the host immune responses. The possibility that CyaA may also affect tissue cells such as respiratory epithelial cells and may be involved in the pathogenesis of the bacterial infection is also indicated.     

Pressure changes induced by whole body acceleration shocks

In the present paper pressure changes induced by sudden body acceleration are studied "in vivo" on the dog and compared to the results obtainable with a recently developed mathematical model. A dog was fixed to a movable table, which was accelerated by a compressed air piston for less than 1 s. Acceleration was varied by changing the air pressure in the piston. Pressure was measured during the experiment at different points along the vascular bed. However, only data obtained in the carotid artery and abdominal aorta are presented here. The results demonstrated that impulse body accelerations cause significant pressure peaks in the vessel examined (about + 25 mmHg in the carotid artery with body acceleration of g/2). Moreover, pressure changes are rapidly damped, with a time constant of about 0.1s. From the present results it may be concluded that, according to the prediction of the mathematical model, body accelerations such as those occurring in normal life can induce pressure changes well beyond the normal pressure value.     

抗心律失常药RP62 71 9对哺乳动物心室肌K~ 电流的抑制

使用全细胞膜片箝技术 ,研究RP6 2 719对内向整流钾电流 (IK1)、瞬时外向钾电流 (Ito)和延迟外向整流钾电流 (IK)的作用 ,并探讨其抗心律失常作用的机制。实验结果表明 ,在指令电压为 - 10 0mV时 ,RP6 2 719可显著抑制豚鼠心室肌细胞IK1,半数抑制浓度 (IC50 )为 5 0± 1 0 μmol/L。RP6 2 71910 μmol/L在 40mV时对犬心室肌细胞Ito抑制率为 84 0± 4 4% ,IC50 为 1 2± 0 5 1μmol/L。在 40mV时 ,5 0 μmol/LRP6 2 719还可使豚鼠心室肌细胞IKstep减少 5 0 0± 8 3% ,IKtail减少 5 6 0± 4 9% ,IC50 分别为 4 2± 0 8μmol/L和 3 3± 0 75 μmol/L。提示RP6 2 719抗心律失常的离子机制与其对IK1、Ito及IK 的抑制有关     

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive moloney sarcoma virus

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C₃-(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.     

Role of lipids in age-related changes in the properties of muscarinic receptors in cultured rat heart myocytes

The effects of the culture's age and of liposome treatments on the properties of muscarinic receptors in cultured rat heart myocytes prepared from the hearts of newborn (1-3 days old) rats were investigated. In these studies we investigated the binding characteristics of antagonists and agonists to the myocyte muscarinic receptors in young (5 days after plating) vs. older (14 days after plating) cultures. Our findings demonstrate that the aging of the cells in culture is accompanied by a reduction in the muscarinic binding capacity and by alterations in the proportion of high- and low-affinity states toward muscarinic agonists, as well as by striking changes in the mode of coupling of the receptors with guanine nucleotide binding protein(s) [G protein(s)]. The above effects of the culture's age occur concomitantly with alterations in the lipid composition of the cultured myocytes (in 14-day old cultures, the phosphatidylcholine/sphingomyelin ratio is reduced, and the cholesterol level is elevated). In order to explore whether the lipid composition is involved in the mechanism that alters the properties and coupling of the muscarinic receptors, we treated aging cultures with liposomes containing egg phosphatidylcholine. This treatment resulted in 14 day old cultures with a lipid composition similar to that of young cultures, and the treated myocytes demonstrated muscarinic receptor properties similar to those of young myocyte cultures. The implications for the role of membrane lipid composition and organization in determining the properties of the muscarinic receptors and their coupling with G proteins are discussed.     

Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes

An important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glycoproteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca++ concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane-stimulus interactions of monocytes as they differentiate into macrophage-like cells, we studied the responses of human monocytes and of monocytes incubated in serum-containing medium for up to 96 hr to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca++ concentrations were maintained during the culture period, intracellular Ca++ levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP-induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum-containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.     

Globular adiponectin activates Akt in cultured myocytes

The serine/threonine kinase Akt plays an important role in insulin-mediated glucose uptake. Adiponectin (Adp) is known to sensitize this process. The purpose of the current study is to investigate if Adp activates Akt independently from insulin; and if Adp synergizes with insulin on Akt phosphorylation in the rat skeletal muscle L6 cells. Differentiated L6 cells were serum-starved and exposed to various concentrations (0-100nM) of recombinant globular Adp (gAdp) and/or insulin for different time periods at 37°C. Phosphorylation of Akt was monitored by Western blot using an antiserum against pSer(473) or pThr(308) Akt. The results demonstrate that gAdp activates Akt in dose- and time-dependent manners. When L6 cells were treated with sub-maximal concentrations of both insulin (10nM) and gAdp (10nM) for 10min neither synergistic nor additive activation of Akt was observed. Similar non-synergistic or non-additive effect of gAdp on insulin-induced Akt activation was also observed in mouse C2C12 myocytes and rat vascular smooth muscle PAC cells. Moreover, pretreatment of the L6 cells with wortmannin (100nM) for 20min significantly reduced gAdp (100nM) induced and insulin (100nM) induced Akt activation by ～80 and ～70%, respectively. These data suggest that adiponectin stimulates Akt activation via the wortmannin sensitive pathway in L6 cells; and that its effects on Akt phosphorylation are not additive to those of insulin.     

Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac myocytes.

Although the prevailing view of mechanoelectric feedback (MEF) in the heart is in terms of longitudinal cell stretch, other mechanical forces are considerable during the cardiac cycle, including intramyocardial pressure and shear stress. Their contribution to MEF is largely unknown. In this study, mechanical stimuli in the form of localized fluid jet pulses were applied to neonatal rat ventricular cells cultured as confluent monolayers. Such pulses result in pressure and shear stresses (but not longitudinal stretch) in the monolayer at the point of impingement. The goal was to determine whether these mechanical stimuli can trigger excitation, initiate a propagated wave, and induce reentry. Cells were stained with the voltage-sensitive dye RH237, and multi-site optical mapping was used to record the spread of electrical activity in isotropic and anisotropic monolayers. Pulses (10 ms) with velocities ranging from 0.3 to 1.8 m/s were applied from a 0.4-mm diameter nozzle located 1 mm above the cell monolayer. Fluid jet pulses resulted in circular wavefronts that propagated radially from the stimulus site. The likelihood for mechanical stimulation was quantified as an average stimulus success rate (ASSR). ASSR increased with jet amplitude and time waited between stimuli and decreased with the application of gadolinium and streptomycin, blockers of stretch-activated channels, but not with nifedipine, a blocker of the L-type Ca channel. Absence of cellular injury was confirmed by smooth propagation maps and propidium iodide stains. In rare instances, the mechanical pulse resulted in the induction of reentrant activity. We conclude that mechanical stimuli other than stretch can evoke action potentials, propagated activity, and reentrant arrhythmia in two-dimensional sheets of cardiac cells.     

Interaction of chemotactic factors with human macrophages: induction of transmembrane potential changes

The electrophysiology of chemotactic factor interaction with cultured human macrophages was investigated with standard intracellular recording techniques. In initial studies, E. coli endotoxin-activated serum, added to cell cultures during intracellular recordings, caused membrane hyperpolarizations which were greater than 30 s in duration, 10-50 mV in amplitude, and associated with decreased membrane resistance. Control serum produced smaller hyperpolarizations lasting 10-20 s and 5-30 m V in amplitude. Endotoxin-activated human serum deficient in the third complement component (C3) did not produce hyperpolarizations unless the serum was reconstituted with C3 before activation. Fractionation of normal activated serum by molecular seive chromatography (G-75 Sephadex) indicated that only fractions that eluted with an estimated molecular weight of 12,500 produced membrane potential changes. The active material that was chemotactic for the macrophages was identified as the small molecular weight cleavage product of C5, C5a, by heat stability (30 min at 56 degrees C) and inactivation by goat antisera to human C5 but not C3. 17 percent of macrophages stimulated with C5a exhibited a biphasic response characterized by a small (2-6 mV), brief (1-10 s) depolarization associated with a decreased membrane resistance preceding the larger and prolonged hyperpolarizations. Magnesium-ethylene glycol bis[β-aminoethyl ether]N,N'-tetraacetic acid (Mg [2.5 mM]-EGTA [5.0 mM]) blocked the C5a-evoked potential changes, whereas colchine (10(- 6)M) and cytochalasin B (3.0 μg/ml did not. Hydrocortisone sodium succinate (0.5 mg/ml) decreased the percentage of cells responding to C5a. In related studies, synthetic N-formyl methionyl peptide (f-met-leu-phe), which had chemotactic activity for cultured macrophages, produced similar membrane potential changes. Repeated exposure of macrophages to C5a or f- met-leu-phe resulted in desensitization to the same stimulus. Simultaneous photomicroscope and intracellular recording studies during macrophage stimulation with chemotactic factor demonstrated that the membrane potential changes preceded membrane spreading, ruffling, and pseudopod formation. These observations demonstrate that ion fluxes associated with membrane potential changes are early events in macrophage activation by chemotactic factors     

抗心律失常药RP62719对哺乳动物心室肌K+电流的抑制

使用全细胞膜片箝技术, 研究RP62719对内向整流钾电流(IK1)、瞬时外向钾电流(Ito)和延迟外向整流钾电流(IK)的作用, 并探讨其抗心律失常作用的机制.实验结果表明, 在指令电压为-100 mV时, RP62719可显著抑制豚鼠心室肌细胞IK1, 半数抑制浓度(IC50)为5.0±1.0 μmol/L.RP62719 10 μmol/L在+40 mV时对犬心室肌细胞Ito抑制率为84.0±4.4%, IC50为1.2±0.51 μmol/L.在+40 mV时, 50 μmol/L RP62719还可使豚鼠心室肌细胞IKstep 减少50.0±8.3%, IKtail减少56.0±4.9%, IC50分别为4.2±0.8 μmol/L和3.3±0.75 μmol/L.提示RP62719抗心律失常的离子机制与其对IK1、Ito及IK的抑制有关.     

Study of membrane potential changes of yeast cells caused by Killer Toxin K1

This work was supported by the NATO Linkage Grant H TECH.LG 930 686.     

Mitochondrial transmembrane potential changes support the concept of mitochondrial heterogeneity during apoptosis.

Dissipation of mitochondrial membrane potential (DeltaPsi(m)) and release of cytochrome c from mitochondria appear to be key events during apoptosis. The precise relationship (cause or consequence) between both is currently unclear. We previously showed in a model of serum-free cultured granulosa explants that cytochrome c is retained in a subset of respiring mitochondria until late in the apoptotic process. In this study we further investigated the issue of heterogeneity by using the DeltaPsi(m)-sensitive probe CM-H2TMRos in combination with a DNA fluorochrome. Changes of DeltaPsi(m) were assessed qualitatively by epifluorescence microscopy and were quantified using digital imaging microscopy. This approach yielded the following results: (a) CM-H2TMRos staining is a reliable and specific procedure to detect DeltaPsi(m) changes in granulosa cells explants; (b) dissipation of transmembrane potential is an early event during apoptosis preceding nuclear changes but is confined to a subpopulation of mitochondria within an individual cell; (c) in frankly apoptotic cells a few polarized mitochondria can be detected. These findings support the hypothesis that ATP needed for completion of the apoptotic cascade can be generated during apoptosis in a subset of respiring mitochondria and is not necessarily derived from anaerobic glycolysis.