Potentiometric Probe Research Articles

Method for reliable detection of different exhaust gas components by pulsed discharge measurements using standard zirconia based sensors

We present a measurement approach to obtain increased differentiation of exhaust gas components and stabilized signal behavior using a standard zirconia-based potentiometric lambda probe. The self-discharge characteristic after different voltage pulses applied to the sensor is used as a measured parameter which depends on gas type and concentration. The detection of NO in the lower ppm range is demonstrated. In addition, this approach allows to improve the response and selectivity to NO significantly compared to other investigated gases as NO2, NH3, H2 and a mixture of hydrocarbons. Therefore, this technique seems to be capable to enable NO sensing in exhaust gas with improved selectivity using standard lambda probes.

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs([1, 2]). Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly([1]). The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods([1]). Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency([1, 3]). In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37 degrees C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS([4]) (5 microMol/L) and briefly illuminated the preparation with 475+/-15 nm excitation light. During a typical 2 second period of illumination, >605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37°C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 μMol/L) and briefly illuminated the preparation with 475±15 nm excitation light. During a typical 2 second period of illumination, >605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

Methodological aspects of measuring absolute values of membrane potential in human cells by flow cytometry

The bis-barbituric acid oxonol, DiBAC(4)(3) is used as a standard potentiometric probe in human cells. However, its fluorescence depends not only on membrane potential but also varies with nonpotential related changes in the amount of intracellular free and bound dye. This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC(4)(3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC(4)(3), the logarithmic relation between cell fluorescence and dye concentration turned into a virtually linear function intersecting with zero. Consequently, calibration can then be confined to determination of the fluorescence of depolarized cells stained with the same concentration as used for the actual measurement of membrane potential. Unexpectedly, quenching of fluorescence occurred in totally depolarized cells at concentrations higher than 6,250 nM. Linearity and quenching could be confirmed by additional experiments on Chinese hamster ovary CHO-K1 and B lymphoblastoid LCL-HO cells.

A comparison of Zn 2+- and Ca 2+-triggered depolarization of liver mitochondria reveals no evidence of Zn 2+-induced permeability transition

Intracellular Zn 2+ toxicity is associated with mitochondrial dysfunction. Zn 2+ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn 2+-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn 2+-induced depolarization with the effects of Ca 2+ in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca 2+ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg 2+, ADP and cyclosporine A. Zn 2+ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn 2+-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca 2+ and Zn 2+ in a calcein-retention assay. Consistent with the well-documented ability of Ca 2+ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn 2+-treated mitochondria. Considered together, our results suggest that Ca 2+ and Zn 2+ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn 2+-induced depolarization, and that Zn 2+ is not a particularly potent mitochondrial inhibitor.

Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p

We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C 3(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C 3(3) by both pumps and confirmed the activation of membrane H +-ATPase by CCCP.

How DASPMI Reveals Mitochondrial Membrane Potential: Fluorescence Decay Kinetics and Steady-State Anisotropy in Living Cells

Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (τ1) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions τ2 and τ3 increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of π-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

TOXI-SIM—A simulation tool for the analysis of mitochondrial and plasma membrane potentials

Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe tetramethylrhodamine methyl ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Δ ψ m) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Δ ψ m and the plasma membrane (Δ ψ p) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Δ ψ m and Δ ψ p over time. Based on Ca 2+ responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces ( http://systemsbiology.rcsi.ie/tmrm/index.html).

Solution pH Alters Mechanical and Electrical Properties of Phosphatidylcholine Membranes: Relation between Interfacial Electrostatics, Intramembrane Potential, and Bending Elasticity

Solution pH affects numerous biological processes and some biological membranes are exposed to extreme pH environments. We utilized micropipette aspiration of giant unilamellar vesicles composed of 1-stearoyl-2-oleoyl-phosphatidylcholine to characterize the effect of solution pH (2–9) on membrane mechanical properties. The elastic area compressibility modulus was unaffected between pH 3 and 9 but was reduced by ∼30% at pH 2. Fluorescence experiments utilizing the phase-sensitive probe Laurdan confirmed gel-phase characteristics at pH 2, explaining the reduction of membrane elasticity. The membrane bending stiffness, k c, increased by ∼40% at pH 4 and pH 9 over the control value at pH 6.5. Electrophoretic mobility measurements indicate that these changes are qualitatively consistent with theoretical models that predict the effect of membrane surface charge density and Debye length on k c, substantiating a coupling between the mechanical and interfacial electrical properties of the membrane. The effect of pH on intramembrane electrical properties was examined by studying the spectral shifts of the potentiometric probe di-8 ANEPPS. The intramembrane (dipole) potential (Ψ d) increased linearly as the solution pH decreased in a manner consistent with the partitioning of hydroxide ions into the membrane. However, changes in Ψ d did not correlate with changes in k c. These mechanical and electrical studies lead to the conclusion that the effect of pH on membrane bending stiffness results from alterations in interfacial, as opposed to intramembrane, electrostatics.

The Na+,K+/H+-antiporter Nha1 influences the plasma membrane potential ofSaccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.

Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8-10 times higher than those measured in blood (75 +/- 38 ng/g wet wt tissue vs. 9.2 +/- 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.

Dominant Role for Calpain in Thromboxane-Induced Neuromicrovascular Endothelial Cytotoxicity

Thromboxane A(2) (TXA(2)) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA(2)-evoked neural microvascular endothelial cell death. Stable TXA(2) mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1alpha,4alpha,5beta(Z),6alpha,(1E,3S)]]-9,11-dedioxy-9alpha,11alpha-methanolpoxy (U-46619) [as well as [1S-[1alpha,2alpha(Z),3beta(1E,3S(*)),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA(2) dependence of these effects was corroborated by antagonism using the selective TXA(2) receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA(2) (mimetics) augmented generation of known active m-calpain (but not mu-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of alpha-spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA(2)-induced neurovascular endothelial cell death.

Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C 3(3) fluorescence assay

Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C 3(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character. Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.

Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S. cerevisiae: effect of pump overproduction and carbon source

The potentiometric fluorescence probe diS-C 3(3) is expelled from S. cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance. The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates. The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained. Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Δψ drop in the late exponential and diauxic growth phase, indicating lowered activity of H +-ATPase. Suppression of activity of both ABC pumps and H +-ATPase obviously signifies cell transition to an energy-saving mode. Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps—a strong dependence of pump activity on the type of the carbon source.

Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

Electrical coupling and plasticity of the mitochondrial network

Kinetic fluorescence imaging and the potentiometric probe tetramethylrhodamine methyl ester (TMRM) were used to evoke and detect changes in membrane potential (δ Ψm) of individual mitochondria in living cells. As a combined effect of preferential TMRM accumulation in mitochondria, and of TMRM photoactivation, individual organelles displayed sharp transient depolarizations caused by local reactive oxygen species (ROS)-mediated gatings of the mitochondrial permeability transition pore (PTP). In COS-7 cells, such directed repetitive gatings of the PTP gave rise to stochastic δ Ψmflickering at the level of individual organelles, but also to prominent synchronous δ Ψmtransitions in whole subgroups of the mitochondrial population, indicative of the existence of an underlying electrically coupled mitochondrial network. In single cells, this network could comprise as much as 65% of the total mitochondrial population, a nd exhibited a high plasticity with mitochondrial units spontaneously connecting to and disconnecting from the coupled structure within seconds. These results indicate that in resting cells, the mitochondrial network is a dynamic proton-conducting struct ure capable to commute and coordinate electrical signals generated by the PTP.

Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore

Confocal laser scanning microscopy and the potentiometric fluorescence probe tetramethylrhodamine ethyl ester were used to measure changes in membrane electrical potential (DeltaPsi(m)) in individual mitochondria after isolation or in the living cell. Recordings averaged over small mitochondrial populations revealed a gradual decline in DeltaPsi(m) caused by the light-induced generation of free radicals. Depolarization was attenuated by dithiothreitol or acidification. In contrast, individual organelles displayed rapid spontaneous depolarizations caused by openings of the mitochondrial permeability transition pore (MTP). Repetitive openings and closings of the pore gave rise to marked fluctuations in DeltaPsi(m) between the fully charged and completely depolarized state. Rapid spontaneous fluctuations in DeltaPsi(m) were observed in mitochondria isolated from rat heart and in mitochondria in living endothelial cells. The loss of DeltaPsi(m) of mitochondria in the living cell coincided with swelling of the organelle and the breakdown of long mitochondrial filaments. In the individual mitochondrion, oxidative stress initially triggered pore openings of shorter duration, before prolonged openings caused the complete dissipation of DeltaPsi(m) and a measurable efflux of larger solutes. Generalizing this scheme, we suggest that under conditions of prolonged oxidative stress and/or cellular Ca(2+) overload, short openings of MTP might serve as an emergency mechanism allowing the partial dissipation of DeltaPsi(m), the fast release of accumulated Ca(2+) ions and the decreased generation of endogenous oxygen radicals. In contrast, loss of matrix metabolites, swelling and other structural damage of the organelle render prolonged openings of the transition pore deleterious to mitochondria and to the cell.

Automated flow injection gradient technique for binding studies of micromolecules to proteins using potentiometric sensors: application to bovine serum albumin with anilinonaphthalenesulfonate probe and drugs.

An automated flow injection (FI) gradient technique is described for the binding study of the potentiometric probe 1-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA). Using a single-channel FI system with a mixing chamber and a flow ANS electrode, the binding parameters (binding constant and number of binding sites) were calculated using the Scatchard model. The concentration gradient was calibrated by injecting ANS in the stream, and the binding experiment was performed by injecting ANS-BSA solution in the carrier solution of equal albumin concentration. The equations describing the concentration gradient and the corresponding electrode potential curve are presented. A systematic study of the factors affecting the complexation equilibrium and the electrode response was performed. For the ANS binding to BSA, two binding classes were determined with binding constants of (2.1 +/- 0.3) x 10(5) and (3.3 +/- 0.8) x 10(3) M-1 and 3.8 +/- 0.6 and 10 +/- 2 binding sites per class, respectively, at 27 +/- 1 degrees C, in 0.10 M phosphate pH 7.4. Competitive binding experiments of sulfamethoxazole, salicylate, azapropazone, ketoprofen, and tolmetin to albumin were also performed by monitoring ANS binding inhibition (decrease of apparent binding constant). This technique takes advantage of FI gradients and direct potentiometry and utilizes the total information contained in FI peaks, providing fast and accurate binding information in a wide range of concentration ratios.

Pharmacological and molecular characterization of ATP-sensitive K + channels in the TE671 human medulloblastoma cell line

ATP-sensitive K + (K ATP) channels in the human medulloblastoma TE671 cell line were characterized by membrane potential assays utilizing a potentiometric fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC 4(3)), and by mRNA analysis. Membrane potential assays showed concentration-dependent and glyburide-sensitive changes in fluorescence upon addition of (−)-cromakalim, pinacidil, diazoxide and P1075. The rank order of potency for these openers was P1075>(−)-cromakalim∼pinacidil>diazoxide. Additionally, glyburide and glipizide inhibited P1075-evoked responses in TE671 cells with half-maximal inhibitory concentrations of 0.22 and 14 μM, respectively. The rank order potencies of both openers and inhibitors were similar to those observed in the rat smooth muscle A-10 cell line. In contrast, in the rat pancreatic insulinoma RIN-m5F cell line, only diazoxide was effective as an opener. Reverse transcription–polymerase chain reaction (RT–PCR) studies detected sulfonylurea receptors SUR2B and SUR1 mRNA in TE671 cells whereas only SUR2B and SUR1 mRNA were, respectively, detected in A-10 and RIN-m5F cells. The inward rectifier Kir6.2 mRNA was detected in all three cell types whereas Kir6.1 was detected only in A-10 cells. Collectively, the molecular and pharmacologic studies suggest that K ATP channels endogenously expressed in TE671 medulloblastoma resemble those present in the smooth muscle.

Aggregation Number of Ionic Surfactants in Complex with Polymer via Measurements of Trace Probe Electrolyte

The distribution of ions in polymer−ionic surfactant solutions was described by the nonlinear Poisson−Boltzmann (PB) theory applied for cell model. On the basis of on the PB cell model, a new trace probe electrolyte method was developed to determine the surfactant aggregation number and the osmotic pressure of the solution. Potentiometric trace probe electrolyte (NaI) measurements were performed for the PVP−NaDS system, and the calculated aggregation numbers were compared to ones measured by fluorescence quenching methods. It was found that the mean aggregation number increases with the surfactant activity and that it is independent of the polymer concentration. The osmotic pressure of the PVP−NaDS system was measured by membrane osmometry.