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.
Evidence suggests that neuronal dysfunction in Huntington's disease (HD) striatum involves deficits in mitochondrial function and in Ca 2+ handling. However, the relationship between mitochondria and Ca 2+ handling has been incompletely studied in intact HD striatal cells. Treatment with histone deacetylase (HDAC) inhibitors reduces cell death in HD models, but the effects of this promising therapy on cellular function are mostly unknown. Here, we use real-time functional imaging of intracellular Ca 2+ and mitochondrial membrane potential to explore the role of in situ HD mitochondria in Ca 2+ handling. Immortalized striatal (ST Hdh ) cells and striatal neurons from transgenic mice, expressing full-length mutant huntingtin (Htt), were used to model HD. We show that (1) active glycolysis in ST Hdh cells occludes the mitochondrial role in Ca 2+ handling as well as the effects of mitochondrial inhibitors, (2) ST Hdh cells and striatal neurons in the absence of glycolysis are critically dependent on oxidative phosphorylation for energy-dependent Ca 2+ handling, (3) expression of full-length mutant Htt is associated with deficits in mitochondrial-dependent Ca 2+ handling that can be ameliorated by treatment with HDAC inhibitors (treatment with trichostatin A or sodium butyrate decreases the proportion of ST Hdh cells losing Ca 2+ homeostasis after Ca 2+ -ionophore challenging, and accelerates the restoration of intracellular Ca 2+ in striatal neurons challenged with NMDA), and (4) neurons with different response patterns to NMDA receptor activation exhibit different average somatic areas and are differentially affected by treatment with HDAC inhibitors, suggesting subpopulation or functional state specificity. These findings indicate that neuroprotection induced by HDAC inhibitors involves more efficient Ca 2+ handling, thus improving the neuronal ability to cope with excitotoxic stimuli.
Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV. Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV. Whereas synthetic and natural ionophores are powerful tools for manipulating cellular physiology, their inherent lack of specificity means that they influence the ion permeability of several membranes in the cell. The two classes of ionophores that are most commonly used are the electroneutral Ca2+/2H+ exchangers typified by ionomycin, and protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2The abbreviations used are: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; [Ca2+]c, cytoplasmic free Ca2+ concentration; ΔΨp, plasma membrane potential; ΔΨm, mitochondrial membrane potential; PMPI, plasma membrane potential indicator; TMRM+, tetramethylrhodamine methyl ester; CGN, cerebellar granule neuron; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; NMDA, N-methyl-d-aspartate; MK801, methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine; TPB-, tetraphenylboron; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino}ethanesulfonic acid; DIV, days in vitro. 2The abbreviations used are: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; [Ca2+]c, cytoplasmic free Ca2+ concentration; ΔΨp, plasma membrane potential; ΔΨm, mitochondrial membrane potential; PMPI, plasma membrane potential indicator; TMRM+, tetramethylrhodamine methyl ester; CGN, cerebellar granule neuron; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide; NMDA, N-methyl-d-aspartate; MK801, methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine; TPB-, tetraphenylboron; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino}ethanesulfonic acid; DIV, days in vitro. Ionomycin is most commonly used with the assumption that it will generate a stable, moderately elevated, cytoplasmic free Ca2+ concentration, [Ca2+]c with maintained cell viability. However, ionomycin also intercalates into the inner mitochondrial membrane where it provides an additional pathway for Ca2+ efflux from the matrix in parallel with the native Ca2+/Na+ antiporter, setting up a proton-dissipating, i.e. uncoupling, Ca2+ cycle that is controlled by the activity of the mitochondrial Ca2+ uniporter and hence by [Ca2+]c (1Åkerman K.E.O. Nicholls D.G. Eur. J. Biochem. 1981; 115: 67-73Crossref PubMed Scopus (78) Google Scholar). The bioenergetic consequences of this are usually ignored, although they could have profound effects on cellular function. Conversely, protonophores such as FCCP that are widely employed to depolarize mitochondria in intact cells can affect plasma membrane potentials at higher concentrations (2Rottenberg H. Wu S.L. Biochim. Biophys. Acta. 1998; 1404: 393-404Crossref PubMed Scopus (223) Google Scholar).An equally important ambiguity surrounds the widespread use of cationic, membrane permeant, fluorescent probes as mitochondrial membrane potential indicators (3Ehrenberg B. Montana V. Wei M.D. Wuskell J.P. Loew L.M. Biophys. J. 1988; 53: 785-794Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 4Farkas D.L. Wei M.D. Febbroriello P. Carson J.H. Loew L.M. Biophys. J. 1989; 56: 1053-1069Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 5Fink C. Morgan F. Loew L.M. Biophys. J. 1998; 75: 1648-1658Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) because their uptake and equilibrium accumulation within the mitochondrial matrix of the cell is responsive equally to the plasma membrane potential, ΔΨp, and the mitochondrial membrane potential, ΔΨm. With the explosion of interest in the multiple roles played by in situ mitochondria in cell physiology and pathology (reviewed in Refs. 6Nicholls D.G. Curr. Mol. Med. 2004; 4: 149-177Crossref PubMed Scopus (224) Google Scholar and 7Duchen M.R. Mol. Aspects Med. 2004; 25: 365-451Crossref PubMed Scopus (568) Google Scholar) has come the importance of accurately monitoring changes in ΔΨm in intact cells. The complicating role of the plasma membrane was recognized at an early stage (4Farkas D.L. Wei M.D. Febbroriello P. Carson J.H. Loew L.M. Biophys. J. 1989; 56: 1053-1069Abstract Full Text PDF PubMed Scopus (208) Google Scholar) and is exacerbated in studies in which both ΔΨm and the plasma membrane potential (ΔΨp) change during the experiment. We have previously presented a semiquantitative technique for the interpretation of whole cell cationic indicator traces under such conditions (8Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar) based on curve fitting and estimates of likely changes in ΔΨp. The approach has proven useful for deciphering experiments in "quench mode" (when the probe concentration in the matrix is sufficient for reversible aggregation) as well as for interpreting traces obtained with probes with differing permeability rate constants (8Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar, 9Rego A.C. Ward M.W. Nicholls D.G. J. Neurosci. 2001; 21: 1893-1901Crossref PubMed Google Scholar, 10Rego A.C. Vesce S. Nicholls D.G. Cell Death Differ. 2001; 8: 995-1003Crossref PubMed Scopus (74) Google Scholar, 11Poppe M. Reimertz C. Düssmann H. Krohn A.J. Luetjens C.M. Böckelmann D. Nieminen A.L. Kögel D. Prehn J.H.M. J. Neurosci. 2001; 21: 4551-4563Crossref PubMed Google Scholar). Quench mode is only applicable to experiments in which rapid step changes in ΔΨm occur while the cell is being imaged (8Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar). Conversely, low probe loadings that avoid matrix quenching must be employed to follow slow changes in potential as well as to estimate pre-existing values of ΔΨm in cell populations (reviewed in Ref. 12Nicholls D.G. Ward M.W. Trends Neurosci. 2000; 23: 166-174Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Under these latter conditions there is serious ambiguity as to whether the observed change in fluorescence is due to a difference in ΔΨp, ΔΨm, or both.It is evidently important to monitor both potentials. Anionic membrane-permeant probes are excluded from polarized cells due to the negative plasma membrane potential but partition increasingly into the cell upon plasma membrane depolarization. Because of their negative charge these probes are not accumulated by mitochondria. Anionic oxonol dyes have been used for several years to monitor changes in ΔΨp (13Civitelli R. Reid I.R. Halstead L.R. Avioli L.V. Hruska K.A. J. Cell. Physiol. 1987; 131: 434-441Crossref PubMed Scopus (32) Google Scholar, 14Apell H.J. Bersch B. Biochim. Biophys. Acta. 1987; 903: 480-494Crossref PubMed Scopus (137) Google Scholar, 15Kiedrowski L. Neuroreport. 2001; 12: 3579-3582Crossref PubMed Scopus (2) Google Scholar, 16Epps D.E. Wolfe M.L. Groppi V. Chem. Phys. Lipids. 1994; 69: 137-150Crossref PubMed Scopus (167) Google Scholar), but their usefulness is compromised by a relatively slow equilibration across the plasma membrane. Recently a proprietary plasma membrane potential assay kit (Molecular Devices, Sunnyvale, CA) has become available in which the problem of background fluorescence from the high extracellular probe concentration is suppressed by a hydrophilic quencher (17Baxter D.F. Kirk M. Garcia A.F. Raimondi A. Holmqvist M.H. Flint K.K. Bojanic D. Distefano P.S. Curtis R. Xie Y. J. Biomol. Screen. 2002; 7: 79-85Crossref PubMed Google Scholar).In this paper a technique was developed for combining the use of the Molecular Devices fluorescent anion (which is termed PMPI, for "plasma membrane potential indicator") with the established cationic indicator tetramethylrhodamine methyl ester (TMRM+). At the same time we have devised a curve-fitting spreadsheet to interpret the traces. As well as providing a more quantitative means of compensating the TMRM+ signal for changes in ΔΨp, this combined technique allows for the first time simultaneous and continuous monitoring of ΔΨp and ΔΨm in cultured neurons. Whereas the technique is validated for cerebellar granule neurons exposed to a variety of ionophores and inhibitors on a confocal microscope, with suitable calibration the methodology is equally applicable to any attached cell preparation and can be used with non-confocal imaging. Finally the curve-fitting spreadsheet may also be used for single-probe studies of ΔΨm, where ΔΨp is invariant or its changes can be estimated.MATERIALS AND METHODSReagents—TMRM+ fluo-4 and fluo-5F were from Molecular Probes (Eugene, OR). PMPI is a proprietary component of the Membrane Potential Assay Kit (R-8042) from Molecular Devices Corp., Sunnyvale, CA. All other reagents were from Sigma.Preparation of Cerebellar Granule Neurons—Cerebellar granule neurons were prepared from 7-day-old Wistar rats as previously described (18Courtney M.J. Nicholls D.G. J. Neurochem. 1992; 59: 983-992Crossref PubMed Scopus (67) Google Scholar) with modifications. Briefly, cells were plated into coverslip-based 8-well chambers (LabTek, Naperville, IL) previously coated with 33 μg/ml polyethyleneimine, at a density of 380,000 cells per 0.8-cm2 well. Cultures were maintained in minimal essential medium supplemented with 10% fetal bovine serum, 30 mm glucose, 20 mm KCl, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. 24 h after plating, 10 μm cytosine arabinoside was added to inhibit growth of non-neuronal cells. Cell cultures were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO2, 95% air and used for experiments at 12–14 days in culture.Plasma Membrane Potential Indicator—An individual vial from a Molecular Devices "membrane potential assay kit, explorer format" (R-8042) containing a proprietary plasma membrane potential indicator was reconstituted in 1 ml of distilled water, dispensed into 50-μl aliquots, and frozen (PMPI stock). For spectral analysis 20 μl of PMPI stock was diluted with 250 μl of water and extracted with 250 μl of octanoyl alcohol to separate the anionic indicator from the aqueous quencher present in the PMPI stock. Excitation and emission spectra (Fig. 1) were determined with a PerkinElmer LS50 scanning spectrophotometer for PMPI in octanoyl alcohol and compared with tetramethylrhodamine in water.Simultaneous Monitoring of PMPI and TMRM+ Fluorescence—Cerebellar granule neurons (CGN) were washed and incubated (37 °C, pH 7.4) for 45 min prior to imaging with a medium (low K-medium) containing 3.5 mm KCl, 120 mm NaCl, 1.3 mm CaCl2, 0.4 mm KH2PO4, 5 mm NaHCO3, 1.2 mm Na2SO4, 15 mm d-glucose, 20 mm Na-TES, 1 μm tetraphenylboron, 5 nm TMRM+, and 0.5 μl/ml PMPI stock. An identical medium in which 120 mm NaCl was substituted by 120 mm KCl was prepared (high-K medium). 5 nm TMRM+ is below the limit for probe aggregation and quenching within the matrix. In some experiments the TMRM+ concentration was varied. The PMPI concentration was chosen to obtain a signal comparable with that of TMRM+. The presence of tetraphenylboron (TPB-) facilitates the equilibration of TMRM+ and other lipophilic cations (4Farkas D.L. Wei M.D. Febbroriello P. Carson J.H. Loew L.M. Biophys. J. 1989; 56: 1053-1069Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 8Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar, 19Scott I.D. Nicholls D.G. Biochem. J. 1980; 186: 21-33Crossref PubMed Scopus (334) Google Scholar) across the plasma membrane. No effect of 1 μm TPB- on the equilibrium distribution of either PMPI or TMRM+ was detected. No extracellular PMPI fluorescence could be detected and addition of 0.5% Triton X-100 abolished both the TMRM+ and PMPI signals associated with the cells.Cells were imaged on a Zeiss Pascal confocal Axiovert 100M microscope equipped with a computer-driven stage. The technique is equally applicable to non-confocal imaging, and unless otherwise stated, the pinhole diameter was increased to give an optical slice of 10 μm to allow collection of the defocused signal from individual somata. Fluorescence was excited in single track mode with the 514-nm band of an argon laser. Because the emission peaks of the two indicators are separated by only 20 nm (Fig. 1) the spectral overlap must be corrected. To accomplish this the emitted epifluorescence was split with a 570-nm dichroic mirror and detected in channel 1 through a Chroma 595–650-nm filter and channel 2 with a Chroma 525–575-nm filter. Crossover between the two channels was quantified with cell preparations loaded with a single probe. The values obtained were dependent upon the amplifier gains for the two channels and so these were kept constant throughout. Laser power was adjusted to obtain an optimal image and did not exceed 5% when used with the ×20 air objective. Whereas the Zeiss software allows for pixel by pixel correction of crossover between the two emission spectra using its subtractive function, it is equally valid to correct single-cell time courses in a spreadsheet. Scan averaging was performed for the high resolution studies shown in Fig. 2 using the ×63 oil immersion objective with line mode summing of four repetitive scans at 1% laser power to minimize phototoxicity.FIGURE 2Confocal images of CGNs equilibrated with PMPI and 5 nm TMRM+. CGNs were equilibrated with PMPI and 5 nm TMRM+ in low- (3.9 mm) K medium as described under "Materials and Methods." Images were corrected for crossover between the fluorophores (Equations 1 and 2). After imaging cells in low-K medium, media was exchanged to increase [K+] to 25 mm and the field was imaged again when re-equilibration was complete. The lower panels show the intensity profiles through the cell, scale in micrometers. Note the reciprocal changes in probe intensity, the lack of co-localization between the PMPI and TMRM+ fluorescence, and the exclusion of PMPI from the nucleus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Curve-fitting Computer Simulation—The simulation to convert the TMRM+ and PMPI fluorescence traces into a time course of changes in ΔΨp and ΔΨm is described in supplementary data where it may be accessed as an Excel spreadsheet. The mathematical background to the simulation is derived below. When applied to data (Figs. 3, 6, 7, 8, and 10) the experimental data points are represented by symbols (closed squares for PMPI, open squares for TMRM+) and the fitted computer simulation by the underlying solid lines. The values for ΔΨp and ΔΨm that are input into the simulation to produce the curve fits are shown in the adjacent graphs.FIGURE 3Calibration of the steady-state fluorescent enhancement of PMPI as a function of ΔΨp; ΔΨp following transient activation of NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (KA) receptors or addition of ouabain. CGNs were equilibrated with PMPI in 3.9 mm K medium as described under "Materials and Methods." A, varying volumes of 120 mm K+ medium were substituted to give final K+ concentrations from 4 to 80 mm with a proportionate decrease in Na+. Plasma membrane potentials were calculated from the Goldman equation assuming conductances for K+, Na+, and Cl- of 1:100:1. (see text). The fluorescent enhancement relative to 4 mm K+ was determined for the mean of 10 cell bodies with no contaminating membrane fragments at each K+ concentration (closed squares, K+ concentration in parentheses). The trend line (solid) is given by a second-order regression curve (see Equation 1). The dotted line represents the fluorescence enhancement that would result from a purely "Nernstian" distribution. The open triangle reports the fluorescent quenching resulting from the hyperpolarization produced by the addition of 0.5 μm valinomycin (in the presence of 5 μg/ml oligomycin). B, where indicated, 100 μm NMDA plus 10 μm glycine (NMDA) was added, followed by 5 μm MK801. Finally the K+ concentration was increased to 25 mm with a proportionate decrease in Na+. C, 175 μm kainate plus 5 μm MK801 were added where indicated, followed by 100 μm NBQX. D, 0.5 mm ouabain. E–G, predicted changes in ΔΨp that produce the best fit between the experimental points in B–D (symbols) and the computer simulation (solid lines in B–D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6The K+-uniport ionophore valinomycin and the K+/H+ antiport ionophore nigericin have opposing effects on CGN ΔΨp and ΔΨm. CGNs were equilibrated in low-K medium with PMPI and TMRM+. A, where indicated by the arrow, 0.5 μm valinomycin plus 5 μg/ml oligomycin was added. Note the transient plasma membrane hyperpolarization and the collapse of ΔΨm as the ionophore clamps the mitochondrial membrane potential to the now negligible K+ gradient across the inner mitochondrial membrane. B, addition of 0.5 μm nigericin to the cells causes a partial depolarization of the plasma membrane followed by a mitochondrial hyperpolarization of 30 mV, consistent with a collapse of a pH gradient of at least 0.5 units across the inner membrane and the compensatory increase in ΔΨm. Unless otherwise stated, this and subsequent experimental traces are the mean of 10 randomly chosen somata with backgrounds subtracted. In this and subsequent figures, the experimental data points are represented as square data points and the fitted computer simulations by the continuous lines. ΔΨp(sim) and ΔΨm(sim), membrane potential time courses that generate the fitted computer simulations in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Glycolysis maintains ΔΨp but lowers ΔΨm. CGNs were incubated in low-K medium with PMPI and TMRM+. A, at the arrow 5 μm myxothiazol was added. The TMRM+ trace is consistent with a depolarization from 150 mm to 124 mV as a result of ATP synthase reversal. The histogram shows the variability in response of individual cell somata. B, 5 μm myxothiaxol plus 5 μg/ml oligomycin were added resulting in a collapse in ΔΨm. Note that ΔΨp is maintained in both cases. The experimental data points are represented as square data points and the fitted computer simulations by the continuous lines. ΔΨp(sim) and ΔΨm(sim), membrane potential time courses that generate the fitted computer simulations in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Low micromolar concentrations of the protonophore FCCP cause a slow plasma membrane depolarization. CGNs were equilibrated in low-K medium with PMPI and TMRM+. Where indicated by the arrows, 5 μg/ml oligomycin together with (A) 0.25 μm FCCP plus or (B) 2.5 μm FCCP were added. Note that the higher concentration of protonophore causes a biphasic depolarization of the plasma membrane. The kinetics of TMRM+ re-equilibration following the high FCCP addition were assumed to be limited by probe redistribution and mitochondrial depolarization was assumed not to be rate-limiting. The resulting rate constant was used for the experiment in Fig. 7A. Each experimental trace is the mean of 10 randomly chosen somata with backgrounds subtracted. The experimental data points are represented as square data points and the fitted computer simulations by the continuous lines. ΔΨp(sim) and ΔΨm(sim), membrane potential time courses that generate the fitted computer simulations in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 10Sequential effects of ionomycin on [Ca2+]c, ΔΨm, and ΔΨp. A, CGNs were preincubated in low-K medium in the presence of 0.5 μm fluo-5F-AM (green) and 5 nm TMRM+ (red). After washing the cells and re-adding low-K medium with TMRM+, 2 μm ionomycin was added. The confocal image was taken 7 min after addition of the ionophore. Note the presence of cells that maintain a low [Ca2+]c and retain mitochondrial TMRM+ (e.g. cell 1), cells that display a high [Ca2+]c but retain TMRM+ (e.g. cell 2), and cells that display a high [Ca2+]c but have lost TMRM+ fluorescence (e.g. cell 3). B, time interval between initiation of the rapid rise in [Ca2+]c (a in C) and the initiation of the decrease in TMRM+ fluorescence (b in C) for single neuronal cell bodies from the experiment depicted in A. C, CGNs were incubated in low-K medium in the presence of PMPI and 5 nm TMRM+. Where indicated 1 μm ionomycin was added and the PMPI and TMRM+ fluorescence time courses were recorded. The experimental data points for a single representative cell body are shown. In parallel, cells loaded with 0.5 μm fluo-5F-AM and 5 nm TMRM+ were exposed to 1μm ionomycin. A cell soma was selected that showed the same kinetics of TMRM+ loss as the first cell and the trace of fluo-5F fluorescence was adjusted on the x axis to synchronize the mitochondrial depolarizations. Note that Ca2+ deregulation precedes the loss of TMRM+. D, computer simulation of changes in ΔΨm and ΔΨp that produce the best fit (solid lines in C) with the experimental traces. Note the biphasic responses of both potentials to ionomycin addition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSFig. 2 depicts equatorial confocal slices through the somata of 12 DIV CGNs equilibrated with PMPI and 5 nm TMRM+. In the polarized cells, PMPI fluorescence is faint and associated with the plasma membrane with slight cytoplasmic fluorescence. Depolarization with 25 mm K+ results in a increase in cell-associated PMPI fluorescence following entry of the probe into the cytoplasm although the probe remains excluded from the nucleus. The decrease in TMRM+ fluorescence is due to the decreased probe accumulation across the plasma membrane in the partially depolarized cells (see Equation 5).Computer Simulation—The computer simulation can be accessed in supplementary data. The theoretical basis of the simulation is developed below.PMPI Calibration—It is first necessary to calibrate the enhancement in the PMPI fluorescence as a function of plasma membrane depolarization. This does not follow an ideal Nernstian relationship, but instead is determined empirically by quantifying the fluorescent enhancement obtained when ΔΨp is depolarized by increasing KCl concentrations. The relationship between ΔΨp and the K+ concentration of the medium (Fig. 3A) was calculated from electrophysiological data in Laritzen et al. (20Lauritzen I. Zanzouri M. Honoré E. Duprat F. Ehrengruber M.U. Lazdunski M. Patel A.J. J. Biol. Chem. 2003; 278: 32068-32076Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) applying the Goldman-Hodgkin-Katz equation. An initial value for the ΔΨp of 8–12 DIV rat CGNs in 3.9 mm KCl media of -83 mV at 37 °C was calculated. A series of experiments were then performed with PMPI-equilibrated CGNs in which step increases in K+ concentrations were made by removing defined volumes of low-K medium and replacing this with an equal volume of high-K medium to give final K+ concentrations from 4 to 80 mm. The mean fluorescent enhancement for 10 cell bodies was determined for each K+ concentration and plotted as a function of ΔΨp (Fig. 4A). The best fit with the empirical fluorescent enhancement was obtained with a second-order regression curve, E=1+0.03(ΔΨKCl−83)+0.0005(ΔΨKCl−83)2 (Eq. 1) where E is the fluorescent enhancement relative to cells in 3.9 mm KCl medium and ΔΨKCl the calculated plasma membrane potential at a given KCl concentration. It should be noted that the fluorescent enhancement is considerably less than that predicted for the change in free cytoplasmic PMPI concentration from the Nernst equation (dashed line in Fig. 3A), presumably due to complicating factors of probe binding to membranes and proteins and changes in fluorescence yield. Using this empirical relationship, an observed change in whole cell PMPI fluorescence can be calibrated as a function of ΔΨp.FIGURE 4Estimation of the volume fraction of the CGN cell soma occupied by the mitochondrial matrix and the quench limit for TMRM+ in the mitochondrial matrix. A, the curve shows the theoretical ratio for the whole cell fluorescence of neurons with depolarized versus polarized mitochondria (Tdepol/Tpol) as a function of the fractional volume occupied by the mitochondrial matrices (x), see Equation 6. Neurons were equilibrated with 5 nm TMRM+ in low-K medium and cell soma were imaged. Myxothiazol (5 μm) plus oligomycin (5 μg/ml) were then added and the final fluorescence determined. PMPI fluorescence confirmed that no change occurred in ΔΨp (see Fig. 7). The vertical lines show the mean (solid line) ± S.E. (dashed lines) for Tdepol/Tpol for 10 randomly chosen cells. The horizontal lines are the corresponding extrapolations to the y axis, indicating a mean matrix fractional volume of 0.023 ± 0.02. B, cells were equilibrated in low-K medium for 60 min with the indicated concentrations of TMRM+ in the absence of TPB-. At the arrow a combination of 2.5 μm rotenone, 2.5 μg/ml oligomycin, and 250 nm FCCP was added to collapse ΔΨm. The transient spike indicates dequenching as aggregated probe is released from the matrix. Each experimental trace is the mean of 10 randomly chosen somata with backgrounds subtracted. C, simulation of experimental traces utilizing a best-fit quench limit of 140 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Single-channel Monitoring of ΔΨp—The PMPI probe may be used in the absence of TMRM+ and tetraphenylboron to monitor changes in ΔΨp using the empirical curve fit in Equation 1. In th
SHORT NOTICES Christ in Context, Divine Purpose and Human Possibility. By EUGENE TESELLE. Pp. xiv+178. Philadelphia: Fortress Press, 1975. $10.95. DAVID NICHOLLS DAVID NICHOLLS Search for other works by this author on: Oxford Academic Google Scholar The Journal of Theological Studies, Volume XXVIII, Issue 1, April 1977, Pages 279-b–280, https://doi.org/10.1093/jts/XXVIII.1.279-b Published: 01 April 1977
Nanomolar concentrations of charybdotoxin or dendrotoxin increase the cytoplasmic free Ca 2+ concentration in isolated central nerve terminals. The effects of the two toxins, normally considered to be blockers of K + channels controlled by voltage in a Ca 2+ ‐sensitive or ‐insensitive manner, respectively, show only marginal additivity. Apamin, an inhibitor of low conductance Ca 2+ ‐activated K + channels, was without effect in either the absence or presence of dendrotoxin. The effect of charybdotoxin on polarized, isolated central nerve terminals seems to be mediated largely by a block of K + channels sensitive to dendrotoxin. Apparently, these voltage‐operated K + channels make a more significant contribution to maintaining the polarized potential of synaptosomes than do those activated by Ca 2+ .
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