Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential: Evidence for interactions between carbocyanine dyes and lipophilic anions

The accumulation of [³H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3′-diethylthiadicarbocyanine iodide and by 3,3′-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.     

Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells

Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.     

Membrane potential of Plasmodium-infected erythrocytes

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.     

Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide

The killing of cultured hepatocytes by tert-butyl hydroperoxide (TBHP) occurs by different mechanisms depending on the presence or absence of the antioxidant N,N'-diphenylphenylenediamine (DPPD). In either situation there is evidence of mitochondrial damage. The mitochondrial inner membrane potential is lost, a result determined by the release from the cells of the lipophilic cation [3H]triphenylmethylphosphonium (TPMP+). Deenergization of the mitochondria is accompanied by a loss of ATP. Oligomycin reduced ATP stores without release of TPMP+ or without effect on the viability of the hepatocytes over the same time course that TBHP killed the majority of the cells. Monensin, a H+/Na+ ionophore, potentiated the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. By contrast, extracellular acidosis reduced the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. Neither monensin nor extracellular acidosis affected the metabolism of tert-butyl hydroperoxide, the release of TPMP+, or the extent of the peroxidation of cellular lipids. These data document the presence of mitochondrial damage in hepatocytes intoxicated with TBHP in both the presence and absence of DPPD. Furthermore, the potentiation by monensin is readily explained by the proposal that mitochondrial deenergization is accompanied by an intracellular acidosis. Such acidosis tends to delay the development of lethal cell injury. The protective effect of extracellular acidosis supports this interpretation.     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

On the composition and characteristics of microvessels isolated from the rabbit and bovine brain

Brain capillaries (microvessels) were isolated from the rabbit and bovine brain. Extensive morphological examinations were performed at the light and electron microscopical levels. The relative contribution of endothelium (52%), basal membrane (32%) and pericytes (16%) to the composition of the microvessel was assessed. The ability of the endothelium from bovine brains to maintain a membrane potential, i.e. to accumulate the lipophilic cation [³H]TPMP, was shown. The transmitter catabolizing enzymes MAO and AchE were shown to be, and COMT and GABA-T not to be associated with the microvessel fraction isolated from rabbits.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Electrogenic nature of lysosomal proton pump as revealed with a cyanine dye

Studies were carried out on the electrogenicity of the lysosomal proton pump using dipropylthiadicarbocyanine iodide (diS-C3-(5] as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C3-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K+ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethylphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluorescein isothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATP-induced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate delta pH across the lysosomal membrane also enhanced the Mg-ATP-induced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K+ medium, mostly due to the K+-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [3H]TPMP and [14C]SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.     

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     

Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ

A rapid cellular-fractionation technique [ Hoek , Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.     

Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes.

Transmembrane electrical and pH gradients have been measured across human erythrocytes and peripheral blood lymphocytes using equilibrium distributions of radioactively labelled lipophilic ions, and of weak acids and weak bases, respectively. The distributions of methylamine, trimethylamine, acetic acid and trimethylacetic acid give calculated transmembrane pH gradients (pHe-pHi) for erythrocytes of between 0.14-0.21 for extracellular pH values of 7.28-7.16. The distributions of trimethylacetic acid. DMO and trimethylamine were determined for lymphocytes, establishing upper and lower limits of the calculated pH gradient over the external pH range of 6.7 to 7.7. Tritiated triphenylmethyl phosphonium ion (TPMP) and 14C-thiocyanate ion (SCN) equilibrium distributions were measured in order to calculate transmembrane electrical potentials, using tetraphenylboron as a catalyst to facilitate TPMP equilibrium. Transmembrane potentials of -7 to -10 mV were calculated from SCN and TPMP, respectively for red cells, and -35 to -52 mV respectively, in the case of lymphocytes. Distributions of TPMP and potassium ions were determined in the presence of valinomycin over a wide range of extracellular potassium concentrations for red cells and the calculated Nernst potentials for TPMP compared to the calculated potential using the Goldman equation for chloride and potassium ions. Distributions of TPMP, SCN and potassium ions were also determined for lymphocyte suspensions as a function of extracellular potassium and the calculated Nernst potentials for TPMP and SCN compared to the calculated potassium diffusion potential.     

Membrane potential changes during IgE-mediated histamine release from rat basophilic leukemia cells

The membrane potential of rat basophilic leukemia cells (RBL-2H3 cell line) has been determined by monitoring the distribution of the lipophilic [3H] tetraphenylphosphonium cation between the cells and the extracellular medium. By this method, the determined potential of these cells, passively sensitized with IgE, is -93 +/- 5 mV (mean +/- SEM, interior negative). Almost 40% of this membrane potential is rapidly collapsed upon the addition of the proton carrier, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). It is suggested that the FCCP-sensitive fraction of the total membrane potential results from the accumulation of this cation by the mitochondria, which maintains a negative membrane potential. Thus, the resting plasma membrane potential of these cells equals -55 +/- 6 mV. During the process of immunological stimulation by antibodies directed against cell membrane bound IgE, the membrane potential decreases. Moreover, there is a correlation between the extent of degranulation of the cells and the depolarization. It is concluded that in common with other secretory systems, depolarization of the plasma membrane is involved in the stimulus-secretion coupling of the histamine secreting RBL cells.     

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.     

The relationship between valinomycin-induced alterations in membrane phospholipid fatty acid turnover, membrane potential, and cell volume in the human erythrocyte

The relationship between alterations in transmembrane potential, cell volume, and phospholipid fatty acid turnover has been examined in human erythrocytes by treating the cells with the monovalent cation ionophore valinomycin. Valinomycin increases the cellular uptake of tetra[3H]phenylphosphonium ion by erythrocytes, indicating membrane hyperpolarization, and causes net loss of potassium chloride and water from the cells leading to a decrease in cell volume. Treatment of erythrocytes with valinomycin also enhances incorporation of [9, 10-(3)H]oleic acid into phospholipids, primarily diacylphosphatidylethanolamine. After replacing intracellular chloride with sulfate and treating cells with the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate, exposure to valinomycin results in uptake of tetra[3H]phenylphosphonium ion and stimulation of [9, 10-(3)H]oleic acid incorporation, but, because anion efflux is prevented, no decrease in cell volume occurs. When tetra[3H]phenylphosphonium ion uptake is also prevented by suspending these cells in 125 mM KCl to dissipate the transmembrane potassium gradient, valinomycin still enhances [9, 10-(3)H] oleic acid incorporation into phospholipid. These results suggest that the presence of valinomycin in the membrane directly alters phospholipid fatty acid turnover and that some of the effects of this ionophore on cellular function previously attributed to alterations in transmembrane potential or cellular potassium content may instead be due to altered phospholipid turnover. Since it is possible that valinomycin may directly perturb phospholipid fatty acid turnover in other cells, the possibility that valinomycin-induced alterations in cellular function are due to altered phospholipid turnover rather than membrane hyperpolarization or altered potassium content should be considered in the interpretation of studies employing this ionophore.     

Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.     

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.     

Effects of medium amino acids on ouabain-sensitive 86Rb+ -uptake and membrane-potential dependent [3H]tetraphenylphosphonium accumulation in Friend erythroleukemia cells

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.     

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.