Plasma membrane potential of neutrophils generated by the Na+ pump.

The plasma membrane potential of human neutrophils was monitored using the anionic dye oxonol-V. The cells maintain a potential of -75 +/- 17 mV when suspended in physiological saline solutions. The cells are scarcely depolarized by extracellular K+ and the depolarization induced by the chemotactic peptide fMet-Leu-Phe is of similar magnitude for cells suspended in 5 or 155 mM K+. Neutrophils are, however, depolarized by suspension in K+-free media or after treatment with ouabain. Neutrophils catalyse Na+-H+ exchange and possess other electroneutral ion transport systems. We propose that the neutrophil membrane potential is generated by an electrogenic Na+ pump, that osmotic stability is achieved by electroneutral ion transport systems and that electrical stability is maintained by anion leakage. Similar mechanisms may also operate in other biological membranes.

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo.

The equilibrium oxidation-reduction mipoint potential (Em) of isolated Rhodopseudomonas sphaeroides cytochrome c2 exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197-208). However, as with mammalian cytochrome c (Brandt, K.G. Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180-4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the Em of cytochrome c2 before and after the conformational change. The Em of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium Em is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c2 to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent Em from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c2 in vivo involve protons.

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.

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria.

The reponses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the 'light-minus-dark' difference spectrum of the chromatophores. The oxonols appear to respond to the light-induced 'energization' by shifting their absorption maxima. In the presence of K+, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes, respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift. The dye response has an apparent second-order rate constant of approx. 2 . 10(6) M-1 . s-1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not possess intrinsic probes of potential.

Sequential onset of permeability changes in mouse ascites cells induced by Sendai virus.

The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. Depending on virus: cell ratio, Ca2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. A model for the induction of permeability changes is presented.

Electrogenic events in the ubiquinone-cytochrome b/c2 oxidoreductase of Rhodopseudomonas sphaeroides.

The reductant of ferricytochrome c2 in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c2 by ZH2 is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c2 reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH2 and ferricytochrome c2, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c2 reduction at pH 7.0 and at 24 degrees C was 2 - 10(9) M-1 - s-1, but this varied with pH, being 5.1 - 10(8) M-1 = s-1 at pH 5.2 and 4.3 - 10(9) M-1 - s-1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.

Effects of ATP and magnesium ions on the fluorescence of harmala alkaloids. Restrictions for the use of harmala alkaloids as fluorescent probes for (Na+ + K+)-ATPase.

1. Harmine and harmaline were investigated as potentially useful fluorescent inhibitors of (Na+ + K+) activated ATPase. 29 From spectroscopic measurements both compounds were shown to form 1 : 1 complexes with ATP, the dissociation constants being 0.65 mM and 1.83 mM for harmine and harmaline respectively. Addition of Mg2+ and enzyme further affected these equilibria. 3. Although it was possible to demonstrate a competitive effect of harmine at the sodium-loading site of the enzyme, other inhibitory effects, including inhibitions of ouabain binding and the ouabain-insensitive ATPase were found. 4. It was concluded that the harmala alkaloids can inhibit (Na+ + K+)-activated ATPase in a complex way involving both Na- and ATP-binding sites. This severely limits their usefulness as spectroscopic probes.

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions.

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystalline-liquid crystalline phase transitions in phospholipid vesicles of dimyristoly and dipalmitoly phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidylcholine show lateral phase separation and in this system the probes parition preferentially into the more 'fluid' phase. In systems that have been reported to contain 'short range order' or 'liquid clustering', such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100.

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.

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.

Understanding the tumor metabolic phenotype in the genomic era.

Now, at the beginning of a new century, 80 years after Warburg's Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.

Characterization of non-lytic cytolysin-membrane intermediates.

In order to understand the nature of cytolysin-membrane interactions, the characteristics of stable, non-lytic cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K562) tumor targets formed cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for cytolysin binding.

Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+.

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.

Rapid switching of ion current in narrow pores: implications for biological ion channels.

Ions flowing through purely synthetic filters made of polyethylene terephthalate which have been etched to produce narrow pores show: (i) rapid transitions between a high-conducting and a low-conducting state; (ii) selectivity of ion flow; and (iii) inhibition by divalent cations and protons. These features resemble those displayed by many biological ion channels. We interpret our results in terms of the special properties of ion conductance at an interface that may be observed whenever the contribution of bulk conductance is minimal.

Time resolved 3-dimensional recording of redox ratio during spreading depression in gerbil brain.

Optical fluorescence and reflectance measurements have been used to map the distribution of metabolic states in three dimensions in the gerbil brain with a spatial resolution of 200 microns an a time resolution of 4-6 s. In Mongolian gerbils anesthetized with pentobarbital, the redox states of the nicotinamide adenine dinucleotide (NADH) and flavoprotein components of the electron transport chain exhibit two distinct phases during the wave of spreading depression: (1) a transient period of oxidation and (2) a prolonged period of reduction, during which the cytochromes are reduced, and the hemoglobin is predominantly in the deoxy form. These data are interpreted as indicating that the energy demand placed on the gerbil brain during such spreading depression wave is sufficient to drive the brain temporarily hypoxic.

Thermal control of drug release by a responsive ion track membrane observed by radio tracer flow dialysis.

The combination of a responsive hydrogel with a rigid porous supporting structure yield a membrane with high mechanical strength and high on-off-permeability ratio. A membrane consisting of an ion track filter with a thermally responsive lining was prepared by penetrating a 19 micron thick foil of poly(ethylene terephthalate) (PET) with swift heavy ions at a fluence of 5 x 10(5) ions/cm2, followed by etching of the ion tracks to generate an ion track filter with 2.9 micron pore diameter, onto which a thin layer of poly(N-isopropylacrylamide) (NIPAAm) hydrogel was grafted. It was revealed that the mass flow of various molecules (water, chloride-, choline+, insulin, and albumin) through the membrane could be thermally controlled. The on-off-permeability ratio ranged between 3 and 10 increasing with molecular weight. Over a storage time of 5 months the permeabilities varied up to a factor of 2.6, while the on-off-permeability ratio and temperature sensitivity remained practically constant.

Plasma membrane potential of lymphocytes from ataxia telangiectasia patients.

The plasma membrane potential of lymphocytes prepared from ataxia telangiectasia (AT) patients and normal subjects was assessed using the optical indicator bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethineoxonol (oxonol-V). AT lymphocytes had a potential of -46 +/- 9 mV and normal lymphocytes had a potential of -63 +/- 4 mV. The intracellular cation content (Na+ and K+) of AT and normal lymphocytes was similar. AT and normal lymphocytes were both depolarized by extracellular K+ and to a similar extent. This study indicates that one feature characterizing ataxia telangiectasia is a modification of the ability of the lymphocyte cell membrane to sustain a normal membrane potential.

Pore-forming toxins: experiments with S. aureus alpha-toxin, C. perfringens theta-toxin and E. coli haemolysin in lipid bilayers, liposomes and intact cells.

Three quite different bacterial toxins (S. aureus alpha-toxin, C. perfringens theta-toxin and E. coli haemolysin) induce the leakage of phosphorylated metabolites from Lettre cells and of calcein from liposomes; in each case leakage is inhibited by Zn2+ greater than Ca2+ greater than Mg2+. Inhibition is not due to displacement of toxin from the membrane, since divalent cations inhibit leakage through pre-formed pores. Electrical conductivity across phospholipid bilayers is induced by each of the three toxins; in each case the probability of channels being in the open state is reduced by divalent cations. Although the pores induced in phospholipid bilayers and liposomes vary greatly in size (theta-toxin much greater than haemolysin greater than alpha-toxin), in Lettre cells the lesions appear more uniform, suggestive of a limiting effect in cells.

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(+)> Zn(2+)>Ca(2+)>Mg(2+). 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.

Mechanisms of attack and defence at the cell surface: the use of phospholipid bilayers as models for cell membrane.

Electrical conductivity across phospholipid bilayers induced by various cytotoxic proteins has been used to analyse the damaging action of such proteins on cells; the protective effect of divalent cations and protons against such attack has also been investigated. The predominant effect of divalent cations and protons is to promote the closed state of membrane pores, i.e. to "gate" protein-induced lesions.