Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Uptake and effects of several cationic dyes on yeast

Summary Several cationic dyes were found to behave as inhibitors of K⁺ uptake in yeast. When added at high concentrations or in a K⁺-free medium, dyes can also produce and efflux of K⁺. The dyes are taken up by the cells in a process that, in different degrees, for several cations requires glucose and is inhibited to a higher degree by K⁺ than by Na⁺.The inhibition of cation uptake is of the competitive type with EB and close to this type with other dyes. Ca²⁺ inhibits the uptake and effects of dyes and in some cases also seems to change the inhibition kinetics on Rb⁺ uptake closer to a pure competitive type.According to preliminary experiments, the efflux of K⁺ seems to be of the electrogenic type, and not due to the disruption of the cells. The data indicate that, independently of the existence of other types of interaction (which do exist), dyes seem to interact with the system for monovalent cation uptake of yeast in different degrees of specificity and energy requirement. This interaction can be followed by fluorescence or metachromatic changes or reduction of the dyes as observed in the dual wavelength spectrophotometer and can be inhibited specifically by K⁺, but not by Na⁺.     

The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

Use of [3H]Triphenylmethylphosphomum Cation for Estimating Membrane Potential in Neuroblastoma Cells

The lipophilic permeant cation [3H]triphenylmethylphosphonium (TPMP) was used to estimate membrane potential in neuroblastoma N1E 115 cells under carefully controlled conditions. The cation distributes into the cells only in the presence of a lipophilic anion, and tetraphenylboron and picrate have been used for this purpose. The potassium salt of tetraphenylboron is poorly soluble, so that studies in high [K+] media are difficult with this anion whereas picrate, at the concentrations required, hyperpolarises the cells. The effect of muscarinic receptor activation was investigated by treating cells with carbachol but no effect was seen either on [3H] TPMP distribution or electrophysiological parameters. The use of [3H]TPMP for qualitative and quantitative evaluation of membrane potential in these cells is discussed.     

Mitochondrial oscillation and activation of H+/cation exchange

Mitochondria incubated aerobically in the presence of tetrapropylammonium and weak acids and in the presence of trace amounts of tetraphenylboron undergo a series of damped oscillations reflecting cycles of osmotic swelling and shrinkage. The matrix volume changes are consequent to transport of tetrapropylammonium catalytically stimulated by tetraphenylboron. The amplitude and frequency of the oscillations increase with the concentration of tetrapropylammonium, as required for critical rates and extents of ion influx. Addition of bovine serum albumin abolishes both the uptake of tetrapropylammonium and the oscillations. Volume oscillations are paralleled by cyclic activation and depression of the respiratory rate. Two lines of evidence suggest that the train of damped oscillations depends on the cyclic activation of an electroneutral exchange of H⁺ with organic cations rather than on cyclic uncoupling. First, further increase of cation permeability due to a pulse of tetraphenylboron, after initiation of cation efflux, restores cation influx. Second, addition of Mg²⁺, which abolishes the oscillations, has a much more marked inhibitory effect on the process of cation efflux than on cation influx. Conversely, addition of A23187, which removes membrane-bound Mg²⁺, promotes cation efflux and thus the oscillations. It is suggested that, in the present system, stretching of the inner membrane and Mg²⁺ depletion result in activation of an electroneutral H⁺/organic cation exchange, and that cyclic activation of this reaction results in damped oscillations.     

Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3-diethylthiodicarbocyanine iodide (DiS-C₂–(5)) and 3,3-dipropylthiodicarbocyanine iodide (DiS-C₃–(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The was found to be-55 mV with two other methods, the distribution of ²⁰⁴T1⁺ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg²⁺-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.Abbreviations ANS aminonaphthalene sulfonate - DiS-C₂–(5) 3,3-diethylthiodicarbocyanine iodide - DiS-C₃–(5) 3,3-dipropylthiodicarbocyanine iodide - EDAC 1-ethyl-3-C-3dimethylaminopropylcarbodiimide - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - MES morpholinoethylsulfonic acid - TPP⁺ tetraphenylphoshonium - TPMP triphenylmethylphosphonium - Tris tris(hydroxymethyl)aminomethane     

Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescence of the dye 3,3-diethylthiodicarbocyanine iodide, and by determining the uptake of the lipophilic triphenylmethylphosphonium cation. The vacuoles have a membrane potential, internal negative, which is estimated to be around-60 mV. These potentials become less negative by nearly 10 mV on addition of ATP. This ATP-dependent depolarisation is inhibited by the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and by the ATPase inhibitors, N,N-dicyclohexylcarbodiimide and trimethyltin chloride, but it is largely insensitive to sodium orthovanadate. Fusicoccin had no significant effect on the isolated vacuoles, but its addition to excised tissue caused a hyperpolarisation of the cells measured using a microelectrode.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DiS-C₂-(5) 3,3-diethylthiodicarbocyanine iodide - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

Cl− transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl^? transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl^? into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl^? uptake and medium osmolarity and (2) complete release of accumulated Cl^? by treatment with detergent. The rate of Cl^? uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl^? was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA⁺), uptake of Cl^? was minimal; transport was stimulated equally by the substitution of either K⁺ or Na⁺ for TMA⁺. Valinomycin in the presence of K⁺ enhanced further Cl^? uptake, while amiloride reduced Na⁺-stimulated Cl^? uptake towards the minimal level observed with TMA⁺. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na⁺ and K⁺; (2) the membrane permeability to the medium counterion determines the rate of Cl^? uptake; (3) Cl^? transport is not specifically coupled with either Na⁺ or K⁺; and, finally (4) Cl^? crosses the tracheal luminal membrane via an electrogenic transport mechanism.     

Interaction of phosphate with monovalent cation uptake in yeast

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb⁺ uptake shows saturation kinetics and the phosphate concentration at which halfmaximal inhibition is observed is equal to the K_m of phosphate for the sodiumindependent phosphate uptake mechanism. The kinetic coefficients of Rb⁺ and Tl⁺ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased.In the case of Na⁺ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na⁺ uptake via a sodium-phosphate cotransport mechanism.Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimenthylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium eptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

The effect of the fluorescent probe, 3,3′-dipropylthiodicarbocyanine iodide,on the membrane potential of Ehrlich ascites tumor cells

The effects of the potential-sensitive fluorescent dye, 3,3′dipropylthiodicarbocyanine iodide, on factors establishing the membrane potential of Ehrlich ascites tumor cells have been tested. The dye itself induces membrane hyperpolarization as monitored by electrophysiological methods. In addition, the dye inhibits active (Na⁺+K⁺-transport and increases cell membrane permeability to K⁺ by about 65% in these cells.     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

Comparison of indirect probes of membrane potential utilized in studies of human neutrophils

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP⁺), 3,3′dipentyloxacarbocyanine [di-O-C₅(3)], 3,3′ dipentylindocarbocyanine [di-I-C₅(3)], and 3,3′ dipropylthiodicarbocyanine [di-S-C₃(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C₃(5) not only exhibits a different fluorescence response mechanism from di-O-C₅(3) [and di-I-C₅(3)] but also that the fluorescence of di-S-C₃(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP⁺ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB^?, to shorten the TPMP⁺ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP⁺/TPB^? are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C₅(3) and di-I-C₅(3) but cannot be avoided with di-S-C₃(5) or TPMP⁺/TPB^?. Using di-O-C₅(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Control of the chemotactic behavior of Bacillus subtilis cells

The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-starved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells.Abbreviations KT medium potassium taxis medium - NAT medium sodium taxis medium - HT medium acidic taxis medium - OHT medium alkaline taxis medium - ImT medium imidazole taxis medium - GT medium glycylgycine taxis medium - Di-S-C₃(5) 3,3-dipropyl-2,2-thiacarbocyanine iodide - TPAs⁺ tetraphenylarsonium ion - TPMP⁺ triphenylmethylphosphonium ion - DDA⁺ dibenzyldimethylammonium ion - TPB^- tetraphenylboron ion - pmf proton-motive force - MCP methyl-accepting chemotaxis protein - MT methylation site - membrane potential     

Anion and amine uptake and uncoupling in submitochondrial particles.

1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KCl or NH4Cl. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-1-napthalene sulphonate cause a pH-independent, 50% uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 muM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two pHases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects; uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as Cl-, the uncoupling effect is independent of Cl-. Furthermore the amine uncoupling is markedly enhanced by lipophilic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.     

Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells

In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocyanine dyes, CC₅ and CC₆, show a unique pattern of intracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing myofilaments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In fibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.     

Visualization of mitochondria and nuclei in living plant cells by the use of a potential-sensitive fluorescent dye

Abstract Cationic potential-sensitive dyes have previously been used to selectively stain mitochondria in living animal cells (Johnson, Walsh & Chen, 1980; Johnson et al., 1981). The present work demonstrates that the cyanine dye 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)) can also be used as a mitochondrial stain in living plant cells. The stained mitochondria were easily visualized by fluorescence microscopy. The accumulation of DiOC₆(3) in mitochondria seemed to be potential-dependent since it was prevented by protonophores, valinomycin and inhibitors of electron transport. It was often observed that DiOC₆(3) also stained the nuclear membrane of some cells. This fluorescence, limited to the perinuclear region, was possibly due to a potential across one or both nuclear membranes, although it was not completely dissipated by any of the ionophores or inhibitors tested. Our observations demonstrate the usefulness of using DiOC₆(3) for studying relative membrane potentials of plant mitochondria and, perhaps, other organelles and membrane systems in living plant cells.