Karel Sigler

SLOW FLUORESCENT INDICATORS OF MEMBRANE POTENTIAL : A SURVEY OF DIFFERENT APPROACHES TO PROBE RESPONSE ANALYSIS

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.

Factors governing substrate-induced generation and extrusion of protons in the yeast Saccharomyces cerevisiae

Experiments with respiration deficient (rho-), ADP/ATP transport deficient (op1) and double (op1 rho-) mutants, with glycolytic and tricarboxylic acid cycle substrates showed that the substrate-induced acidification of yeast suspensions is closely associated with glycolysis. The glucose/proton stoichiometry is 2.5 : 1 to 4 : 1 depending on glucose concentration. The kinetics of the process are complex, the acidification curve having a very fast initial component and two slower exponential components. The first component suggests an initial proton efflux from endogenous sources, triggered by exogenous substrates. The acidification process exhibits two Km values at about 1 and 15 mM D-glucose, indicating two distinct saturable pathways of proton extrusion. The total extent of acidification and thus the final pHout reaches a saturation value with increasing glucose concentration and suspension density. Both the total extent and the rate of acidification are subject to control by extracellular pH which reflects the tendency of the cells to build a fixed [H+]out/[H+]in ratio. When the control is lifted, both quantities are considerably increased. A crucial role in the substrate-induced acidification is thus played by active membrane processes and their control mechanisms.

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H+). The buffering action of passively excreted metabolites (CO2, organic acids) and cell surface polyelectrolytes contributed only 15--40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 nmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence K+ greater than Rb+ much greater than Li+ congruent to Cs+ congruent to Na+. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO2 and organic acids, net extrusion of H+ and K+ (in K+-free media; in K+-containing media this is preceded by an initial rapid K+ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.

Mechanisms of acid extrusion in yeast

II. Su ~strate-induced acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 A. Regulation of proton flux by external pH and buffering capacity . . . . . . . . . . . . . . . . . . . . . . 376 B. Regulation by substrate uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 C. Regulation by cell concentrat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 D Regulat ion by metabolic s tate of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 E Intracellular titratable acidity, during acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

Membrane potential in yeast cells measured by direct and indirect methods

The membrane potential, delta psi, of various yeasts estimated from the distribution of tetraphenylphosphonium cations ranged from -50 to -120 mV, depending on species, incubation conditions and technique of measurement. Values obtained directly with a microelectrode in Endomyces magnusii were consistently lower than those determined indirectly.

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.

The effect of non-electrolyte osmolarity on frog oocytes. I. Volume changes.

Abstract The time course of oocyte volume in mannitol hypertonic media is characterized by a biphasic curve; shrinkage of the cells leads to a volume minimum and this is followed by a slower swelling. Behaviour of this type may be explained by a mathematical model in which the inflow of mannitol is followed by osmotically driven uptake of water. The permeability of the oocyte surface towards mannitol appears to increase with increasing the mannitol concentration in the medium. In hypotonic saline the time course of the oocyte volume is again biphasic; from chemical analysis it may be concluded that the initial swelling is followed by a slower shrinkage mostly due to a loss of osmotically active potassium chloride from the cells. This process may be described by a model taking into account the non-zero hydrostatic pressure inside the oocytes.

Ion transport in the pitcher of Nepenthes henryana

Abstract Ion transport in the pitcher of Nepenthes henryana has been studied: 1. 1. Apparent concentrations of Na+, K+, H+, Ca2+, Mg2+ and Cl− in the pitcher tissue and liquid were determined. 2. 2. By 24-h dialysis of the pitcher liquid against redistilled H2O about 90% of the K+ and Cl− were removed, as compared with only 10% Na+. 3. 3. The apparent membrane potentials for Na+, K+ and Cl- at the interphase pitcher tissue-liquid were calculated according to the Nernst equation. 4. 4. Using microelectrodes, the membrane potential of the cells lining the inner surface of the pitcher was found to be 35 ± 3.7 mV (range 17–55 mV; 48 measurements in a total of 10 plants). The tissue was negative with respect to the pitcher liquid. 5. 5. In Na+-containing media a potential difference of 30–50 mV and a short-circuit current of 10–20 μA/cm2 across the pitcher wall were found. Both of these values were decreased by 0.4 mM 2,4-dinitrophenol or 0.4 mM HgCl2. 6. 6. Na+ in the bathing liquid produced a short-circuit current in the direction: inner compartment → pitcher tissue → outer medium ; K+ and Cl− brought about currents in the opposite direction. 7. 7. The mechanism of the transport of Na+, K+ and Cl+ in the pitcher is discussed.

Some properties of the adenosine triphosphatase systems of two yeast species, saccharomyces cerevisiae and rhodotorula glutinis

Summary1.Total ATPase levels were determined in homogenate fractions of bakers yeast,Saccharomyces cerevisiae K andRhodotorula glutinis. The maximum ATPase activities in 8000 × g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pi h−1 (g DS)−1, respectively; the activities in the sediment were somewhat higher. Exponential cells ofS. cerevisiae K andR. glutinis exhibited higher ATPase levels than did the stationary cells.2.The total ATPase activity in both yeast species showed a maximum at pH 6.8, a minimum at pH 7.2, and another broader maximum around pH 8.0.3.No significant Na,K-ATPase activity was detected in bakers yeast, in either the exponential or the stationary cells ofR. glutinis, and in exponentialS. cerevisiae K cells in the pH range of 6.0–9.0.4.Stationary cells ofS. cerevisiae K exhibited, at pH 7.0–8.5, a Na,K-ATPase activity attaining 9% of total ATPase level.5.3 × 10−3m phenylmethyl sulphonyl fluoride had no effect on the total ATPase level inS. cerevisiae and inhibited the activity inR. glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence.6.1.5m urea lowered the ATPase activity inR. glutinis by 68% but had no effect onS. cerevisiae cells. 10−5m dicyclohexylcarbodiimide suppressed the ATPase activity inS. cerevisiae andR. glutinis by 74 and 79%, respectively. Neither agent revealed any additional Na,K-ATPase activity.7.The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lowest flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions. The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.

The effect of non-electrolyte osmolarity on frog oocytes: II. Intracellular potential

Abstract The intracellular potential of the oocytes of Rana temporaria and Rana esculenta was found to be reduced when the osmolarity of the external medium was increased by various concentrations of mannitol. A model is described in which the permeability coefficients for Na + and K + were calculated from the rates of isotopic exchange by Goldman-Hodgkin-Katz approximations and the phenomenon may be explained by a dependence of the two coefficients and of the intracellular concentrations of the two ions on osmolarity. The state of Na + , K + and Cl − inside the cells as well as the nature and magnitude of the active transport processes across the surface of the oocyte are discussed.