C. Lindsay Bashford

Causes and consequences of tumour acidity and implications for treatment

Tumour cells have a lower extracellular pH (pHe) than normal cells; this is an intrinsic feature of the tumour phenotype, caused by alterations either in acid export from the tumour cells or in clearance of extracellular acid. Low pHe benefits tumour cells because it promotes invasiveness, whereas a high intracellular pH (pHi) gives them a competitive advantage over normal cells for growth. Molecular genetic approaches have revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important mechanism for establishing the metabolic phenotype of tumours. Understanding tumour acidity opens up new opportunities for therapy.

The Behavior of Oxonol Dyes in Phospholipid Dispersions

The interaction of a class of oxonol dyes with sonicated phospholipid vesicles was followed optically. The spectra of vesicle-associated dyes resemble those found for the dyes in organic solvents, indicating that the oxonols occupy a hydrophobic region of the membrane. At equilibrium the affinity of the oxonols for the vesicles depends on the structure of the dye, the physical and chemical composition of the vesicles, and the ionic strength of the medium. The oxonols occupy soybean lipid vesicles to a level of 147.9 +/- 17.1 nmol/mg lipid with a dye membrane dissociation constant of 3.33 +/- 0.54 muM. The interaction of the oxonols with soybean lipid vesicles is biphasic. The fast phase has a second order rate constant of 9.04 +/- 0.36 x 10(6)M(-1) s(-1) and the number of fast binding sites, 68 +/- 8 nmol/mg lipid, was determined from the ratio of the second order rate constants obtained with lipid and with dye in excess. The dissociation of oxonols from soybean lipid vesicles is also biphasic, and the fast process has a rate constant of 17 +/- 2 s(-1), yielding a dissociation constant for the fast sites (k(-1)/k(2)) of 1.88 +/- 0.15 muM. The slow phases of oxonol association with, and release from, soybean lipid vesicles are not second order and have half times of between 0.2 and 5 min, depending on the physical and chemical composition of the membrane lipids. The amplitudes of the slow phases are sensitive to the composition of the aqueous media on each side of the vesicle membranes, which suggests that the slow processes represent the permeation of the membrane by the oxonols. The importance of the properties of the oxonol dyes in the interpretation of their behavior in natural membranes is discussed.

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

SummaryLeakage of ions (Na+, K+) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by ‘pore-forming’ agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca2+, Mn2+ or Ba2+; Mg2+, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca2+) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na+, K+, Rb+, NH4+) and divalent (Mg2+, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn2+ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.

The measurement of membrane potential using optical indicators.

ConclusionsOptical methods have become established as a major experimental protocol for following membrane potential. They can provide a rapid, continuous record of the potential and have a very wide applicability. However, when used to make quantitative assertions about membrane potential, optical methods have a number of weaknesses. Even the most reliable calibration procedures depend on accurate evaluation of a small number, namely the internal ion concentration, in a large background, that is total ion levels. However, a consensus seems to be emerging that the plasma membrane potential of non-excitable cells nevertheless has considerable magnitude: typical values are −60 mV for lymphocytes (Rinket al., 1980), −20 to −100 mV, depending on metabolic load, for Ehrlich ascites tumour cells (Philo & Eddy, 1978; but see also Smith & Robinson, 1980), and −66 to −86 mV for neutrophils (Tathamet al., 1980). In our own experiments using monolayer cultures of cells grown to confluence (Bashfordet al., 1981) the potential across the plasma membrane is of the order of −100 mV (see Fig. 2). Membrane potentials of similar magnitude have been found using ion-distribution methods and microelectrodes in neuroblastoma cells and lymphocytes (Deutschet al., 1979a,b). In the latter studies ions of different charge were used to provide upper and lower estimates of the potential, the presumed effects of binding being very different for anions and cations. A similar approach, in this case the use of optical indicators of different charge, has been taken by Rinket al. (1980), and this would seem to be one way in which to diminish the uncertainties involved in dye calibration. Unfortunately many anions, particularly oxonols, form complexes with valinomycin (Lavie & Sonenberg, 1980; Rinket al., 1980), although we have found no evidence for such a complex with bis isoxazolone oxonols (J.C. Smith and C.L. Bashford, unpublished observations). It is apparent that calibration procedures not dependent on valinomycin should be sought in order to establish optical methods as a quantitative approach to the study of membrane potential.

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.

31P-Magnetic resonance spectroscopy studies of nucleated and non-nucleated erythrocytes; time domain data analysis (VARPRO) incorporating prior knowledge can give information on the binding of ADP

Human erythrocytes have no nucleus, mitochondria or endoplasmic reticulum, whereas chicken erythrocytes have a nucleus and mitochondria and are closer in internal morphology, to cells such as the hepatocyte. Erythrocytes were used to test the hypothesis that 31P-MRS invisibility of ADP is associated with the presence of intracellular organelles. Simple frequency domain spectral analysis methods showed that all the acid extractable ADP (and ATP) was MR-visible in human erythrocytes. However, such methods gave variable estimates for 31P-NMR spectra of fresh chicken erythrocytes from which no conclusions could be drawn about the MR-visibility of ADP. Only when the data were fitted by a method incorporating prior knowledge of the ATP and ADP peak structure, using the time domain VARPRO method, was it possible to conclude that in fresh chicken erythrocytes, similar to other nucleated cells (liver, muscle), all the acid extractable ADP appeared to be MRS invisible, indicating binding or sequestration by intracellular organelles.

Structure-based prediction of the conductance properties of ion channels

The HOLE procedure allows the prediction of the absolute conductance of an ion channel model from its structure. The original prediction method uses an empirically corrected Ohmic method. It is most successful, with predictions being reliable to within a factor of two. A new modification of the procedure is presented in which the self-diffusion coefficients of water molecules from molecular dynamics simulation are used to replace the empirical correction factor. A prediction of the conductance for the porin OmpF by the new method is made and shown to be very close to the experimental value. HOLE also allows the prediction of the effect that the addition of non-electrolyte polymers will have on channel conductance. The method has great potential to yield structural information from data provided by single channel recordings but needs further validation by making measurements on channels of known structure. Preliminary results are given of single channel records establishing the effects of non-electrolytes on the conductance of gramicidin D channels. As an example of the potential uses of the procedure application is made to examine the oligomerization of alpha-toxin (alpha-hemolysin) channels. A model for the alpha-toxin hexamer, based on the crystal structure for the heptamer, is generated using molecular mechanics methods. The compatibility of the structures with single channel conductance data is assessed using HOLE.

The oxidation—reduction state of cytochrome oxidase in freeze trapped gerbil brains

The steady state level of reduction of cytochrome oxidase in purified mitochondrial preparations is usually extremely low [ 1,2] (<S%), the enzyme becoming significantly more reduced only when the oxygen tension of the medium falls below 1 mm Hg [3-51. However, studies of the cat cerebral cortex suggest that in the aerobic steady state in vivo cytochrome oxidase may be up to 85% reduced [6,7]. The haem proteins associated with oxygen delivery and consumption, haemoglobin and mitochondrial cytochromes, can be conveniently monitored by their specific absorbance in the 500-650 nm spectral region using dual-wavelength spectroscopy either in the conventional transmission mode [ 1,2,8] or by reflectance [6,7,9,10]. Here, scanning dual-wavelength reflectance spectroscopy was employed in order to resolve the overlapping absorbance bands of haemoglobin and mitochondrial cytochromes in freezetrapped gerbil brains. In these preparations the steady state level of cytochrome oxidase remains constant over a wide range of inspired oxygen concentrations; the enzyme exhibiting significant reduction only when the fraction of oxygen in the inspired gasses (FiOz) fell below 0.05.

Fluctuation of surface charge in membrane pores.

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.

Membrane pores—From biology to track-etched membranes

Flow of ions through narrow pores, either induced in biological membranes or created in synthetic membrane filters, exhibits, under appropriate conditions: 1) rapid switching of ion current between high and low conducting states; 2) selectivity between different ions; 3) inhibition by protons or divalent cations with an order of efficacy usually H+ >Zn2+>Ca2+ >Mg2+. It seems reasonable to conclude that these common properties arise from a common cause-the nature of the flow of ions close to a charged surface.