Action of 1-fluoro-2,4-dinitrobenzene on passive ion permeability of the human red blood cell

Summary Dinitrofluorobenzene (DNFB) inhibits the penetration of anions such as sulfate, phosphate, succinate, and lactate, and facilitates the penetration of cations such as K⁺ and Na⁺. The phlorizin-glucose insensitive fraction of erythritol permeability is not affected by the agent. The effects of DNFB on ion permeability are similar to those of more specific amino reactive agents like trinitrobenzene sulfonate and 2-methoxy-5-nitrotropone.Anion permeability reacts more sensitively to DNFB than cation permeability. At a given concentration of DNFB in the medium, the inhibition of anion permeability develops faster than the facilitation of cation permeability. At a given time of exposure, lower concentrations of DNFB are required to produce a nearly maximal response of anion permeability than are necessary for maximal effect on cation permeability.The response of anion and cation permeability to DNFB is augmented by increasing the pH at which dinitrophenylation is allowed to take place.DNFB binding to the cell membrane is about one order of magnitude lower than DNFB binding to the whole cell. In the cell membrane, proteins as well as lipids are dinitrophenylated. Among the lipids, only phosphatidylethanolamine binds significant amounts of DNFB. Phosphatidylserine does not seem to react with the agent under the experimental conditions under which DNFB produces its effects on ion permeability.The experimental results are compatible with the assumption that removal of uncharged NH₂-groups by dinitrophenylation of the membrane leads to a concomitant reduction of fixed NH ₃ ⁺ -groups and hence of the positive membrane charge. This leads to an acceleration of cation movements and an inhibition of anion permeability while nonelectrolyte permeability remains unaffected. However, other explanations of our observations cannot be ruled out.     

Influence of ethanol on the high frequency electric polarizability of E. coli

The interface electric polarizability of bacteria (charge dependent (ChD) and Maxwell-Wagner (MW) polarizabilities) gives information about their electric charge, determined by the structure and functional state. It is well known that the polarizability could be changed significantly by adding some substances to the suspension, and can be measured using an electro-optical (EO) method. There are some literature data, according to which the adding of ethanol decreases the electric polarizability of the cells. However the reason for the change in this parameter is not clear, as well as which component (ChD and/or MW) of polarizability has the main contribution. Generally the present work shows that the effect of ethanol is connected to the change of the internal (cytoplasm) MW polarizability and is mainly caused by increasing the cell membrane permeability. This results in an ionic flow through the membrane, which velocity and direction depends on the relative values of the inner (cytoplasm) and the outer medium ionic strength.     

A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis.

Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites

Divalent cations and group-specific chemical modifiers were used to modify sodium efflux in order to probe the molecular structure of sodium channels in dog red blood cells. Hg++, Ni++, Co++, and PCMBS (parachloromercuribenzene sulfonic acid), a sulfhydryl reactive reagent, induce large increases in Na+ permeability and their effects can be described by a curve which assumes 2:1 binding with the sodium channel. The sequence of affinities, as measured by the dissociation constants, reflects the reactivity of these divalent cations with sulfhydryl groups. In addition, the effects of Hg++ and PCMBS can be reversed by the addition of dithiothreitol, an SH-containing compound, to the medium. Much smaller increases in Na+ permeability are produced by Zn++ and the amino-specific reagents, TNBS (2,4,6-trinitrobenzene sulfonic acid) and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid). The Zn++ effect can be described by a curve which assumes bimolecular binding with the channel, and its effect on Na+ permeability can be reversed by the addition of glycine to the medium. The effects of Ni++ and SITS can be completely reversed by washing the cells in 0.16 M NaCl while TNBS binding is partially irreversible. Measurements of mean cell volumes (MCV) indicate that the modifier-induced increases in Na+ permeability are not caused by shrinkage of the cells. It is concluded that the movement of sodium ions through ionic channels in dog red blood cells can be enhanced by modification of amino and sulfhydryl groups. Zn++, TNBS, and SITS increase Na+ permeability by modifying amino groups in the channel while Hg++, Ni++, Co++, and PCMBS act on sulfhydryl groups.     

Pore-forming colicins: synthesis, extracellular release, mode of action, immunity

Colicins are bacterial toxins encoded by plasmids which also confer immunity to producing cells. In a first stage, colicins are synthesized in the cytoplasm of colicinogenic cells. Subsequently they are released into the extracellular medium following the action of a small protein synthesized coordinately with the colicins. This protein is a lipoprotein and causes a non-specific increase in the envelope permeability, in particular, through the activation of an outer membrane phospholipase. After release into the medium, colicins kill sensitive cells in 3 defined steps: adsorption onto a specific receptor at the surface of the bacterium, translocation across the outer membrane and action. A specific domain of the colicin molecule is responsible for each of these steps. The most common colicins are those which kill by depolarizing the cytoplasmic membrane with the formation of voltage-dependent ionic channels. Immunity is conferred to producing cells by a membrane protein which interacts with the colicin and prevents formation or functioning of these ionic channels formed by its C-terminal domain.     

Erythrocyte membrane sulfhydryl groups and cation permeability

Reaction of the slowly penetrating organic mercurial compound parachloromercuribenzene sulfonate (PCMBS) with intact erythrocytes has been characterized. Addition of concentrations of PCMBS which result in binding within the interior of the membrane of more than 1.9 × 10^?18 moles/cell produces alterations in Na⁺ and K⁺ permeability, but does not affect choline permeability. However, the increased cation permeability is observed only after a lag period of over two hours. After ten hours, a spontaneous slow “recovery” to normal rates of K⁺ leakage occurs at 25°C but not at 2°C. Subsequent to the effects on cation balance, increasing degrees of hemolysis occur, interpreted as colloid osmotic lysis. The relationships between the binding of the agent and its effects are as follows: a small, rapid initial uptake does not affect cation permeability; the subsequent slower uptake is associated with increased leakage of K⁺ and Na⁺; and the recovery at 25°C is associated with desorption of about half of the PCMBS due to competition by soluble thiol substances released into the medium from the cells. Desorption and “recovery” can be mimicked at any time by addition of small amounts of protein in the medium. The half of the PCMBS that cannot be desorbed is assumed to be bound by the hemoglobin inside the cell. The sulfhydryl groups involved in control of cation permeability constitute only a fraction of the total within the membrane (4–18%). They are located within the interior of the membrane separated from the medium and from the interior of the cell by diffusion barriers to PCMBS.     

Elimination of permeability mutants from selections for drug resistance in mammalian cells

Chinese hamster ovary (CHO) cells exhibit increased sensitivity to a wide variety of microtubule inhibitory drugs when verapamil is present in the growth medium. The extent of this increased sensitivity is drug specific: some drugs such as taxol and vinblastine respond greatly to the presence of verapamil, whereas other drugs such as griseofulvin respond very poorly. For the majority of drugs examined, however, a 2- to 10-fold increase in drug sensitivity is observed in the presence of verapamil at 5 micrograms/ml. The effects of verapamil are even more dramatic when drug-resistant mutant cells with a presumed alteration in membrane permeability are examined. In the presence of appropriate levels of verapamil, these mutants demonstrate a level of drug sensitivity comparable to that of the wild-type parental cells. Drug-resistant cells from similar selections but with well-defined alterations in alpha- or beta-tubulin and no evidence of alterations in membrane permeability, however, continue to exhibit increased resistance to the selecting drug even in the presence of verapamil. These studies support the conclusion that verapamil affects the membrane permeability to or transport of a wide variety of hydrophobic drugs. In addition, we have used this information to devise selections that virtually eliminate the isolation of drug-resistant permeability mutants. This methodology should be generally applicable to genetic studies of drug action that are complicated by the isolation of large numbers of mutants with permeability alterations.     

The effects of cation-induced and pH-induced membrane stacking on chlorophyll fluorescence decay kinetics

We have compared the effects of thylakoid membrane appression by electrostatic screening and by charge neutralization on the room-temperature chlorophyll fluorescence decay kinetics of broken spinach chloroplasts. Monovalent and divalent metal cations induce both a structural differentiation of thylakoid membranes and a lateral segregation of pigment-protein complexes. These phenomena have distinct effects on the F0- and Fmax-level chlorophyll fluorescence decay kinetics at different levels of added cation. We further find specific cation effects, particularly on a 1-2 ns decay component at the Fmax fluorescence level, that are proposed to be related to the effectiveness of electrostatic screening as determined by the hydrated metal ionic radius. Distinct pH-induced effects on chlorophyll fluorescence decay kinetics are associated with the alternative mechanism of electrostatic neutralization to induce membrane stacking. These observations are used to construct a model of chlorophyll fluorescence emission that accounts for the variable kinetics and multiexponential character of the fluorescence decay upon membrane appression.     

Plasma membrane permeability as an indicator of salt tolerance in plants

There is evidence that the plasma membrane (PM) permeability alterations might be involved in plant salt tolerance. This review presents several lines of evidence demonstrating that PM permeability is correlated with salt tolerance in plants. PM injury and hence changes in permeability in salt sensitive plants is brought about by ionic effects as well as oxidative stress induced by salt imposition. It is documented that salinity enhances lipid peroxidation as well as protein oxidative damage, which in turn induces permeability impairment. PM protection, and thus retained permeability, in tolerant plants under salt imposition could be achieved through increasing antioxidative systems and thereby reducing lipid peroxidation and protein oxidative damage of PM. It appears that specific membrane proteins and/or lipids are constitutive or induced under salinity, which may contribute to maintenance of membrane structure and function in salt tolerant plant species. Furthermore, protecting agents (e.g., glycinebetaine, proline, polyamines, trehalose, sorbitol, mannitol) accumulated in salt tolerant species/cultivars may also contribute to PM stabilization and protection under salinity. Based on the presented evidence that PM permeability correlates with plant salt tolerance, we suggest that PM permeability is an easy and useful parameter for selection of genotypes of agriculture crops adapted to salt stress.     

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K⁺ permeability. A fluorometric assay performed with 3,3′-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K⁺-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.     

Inhibitory effects of newly synthesized sulfonamide derivatives on calmodulin-dependent phosphodiesterase activity and their modulation of the external ATP-dependent permeability change in mammalian cultured cells

External ATP causes a passive permeability change in several types of transformed cells and this change is further enhanced by calmodulin antagonists, such as trifluoperazine. However, such drugs also have nonspecific effects on membrane permeability. We have synthesized several new sulfonamide derivatives, which were found to inhibit calmodulin-dependent phosphodiesterase. The drugs also enhanced the ATP-dependent permeability change in CHO-K1 cells, but their effective concentration ranges were wider than those of previously known antagonists, and thus they would be useful for pharmacological use.     

Drug resistance and membrane alteration in mutants of mammalian cells.

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.     

Ionenkanäle – Einführung aus physiologischer Perspektive

Ion channels form a complex class of membrane transport proteins. They are often classified according to their selective permeability for particular ion species as well as to their gating properties, which are controlled by either membrane voltage, ligand binding or physical stimuli. Ion transport through membrane pores embedded in protein channel complexes possesses both a chemical and an electrical dimension with ion flux causing both charge separations as well as changes in ionic concentrations. This electrochemical double-nature of ion transport is reflected in the two main physiological domains of ion channel function: in excitable cells many ion channels predominately control membrane voltage to generate fast electrical signaling, while epithelial or intracellular ion channels are mainly involved in directional ion transport. Given this framework, individual channelopathies display their major deficiencies either in fast electrical signaling or ion transport itself.     

Permeability of lipid bilayers to water and ionic solutes

The lipid bilayer moiety of biological membranes is considered to be the primary barrier to free diffusion of water and solutes. This conclusion arises from observations of lipid bilayer model membrane systems, which are generally less permeable than biological membranes. However, the nature of the permeability barrier remains unclear, particularly with respect to ionic solutes. For instance, anion permeability is significantly greater than cation permeability, and permeability to proton-hydroxide is orders of magnitude greater than other monovalent inorganic ions. In this review, we first consider bilayer permeability to water and discuss proposed permeation mechanisms which involve transient defects arising from thermal fluctuations. We next consider whether such defects can account for ion permeation, including proton-hydroxide flux. We conclude that at least two varieties of transient defects are required to explain permeation of water and ionic solutes.     

Synergistic incorporation of daunorubicin in erythrocytes in the presence of polyene antibiotics. Role of the membrane potential

The synergistic incorporation into red blood cells of the antitumor compound daunorubicin, in the presence of the polyene antibiotics amphotericin B and vacidin A, depended on the composition of the external medium. Synergism was observed only for concentrations of polyene antibiotics that induce cation permeability. The same synergistic effect was observed with the K⁺ selective carrier, valinomycin, but this had a different dependence on the external medium composition. By using the membrane probe 3,3′-dipropylthiadicarbocyanine (diS-C₃-(5)), the synergistic effect was shown to occur under conditions where addition of the ionophores leads to hyperpolarization of the membrane.     

Leucocyte locomotion and chemotaxis : The influence of divalent cations and cation ionophores

Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca²⁺ and Mg²⁺ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could be reversed by adding low doses of divalent cation ionophores (X537A or A23187) to the Ca²⁺- and Mg²⁺-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracellular divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K⁺ depletion or of ouabain on leucocyte locomotion was noted.     

alpha-Latrotoxin as an instrument for studying neurosecretions

alpha-Latrotoxin, a presynaptic toxin from black widow spider venom Latrodectus mactans tredecimguttatus, triggers exocytosis in a variety of neurosecretory cells both in the presence and absence of calcium in the medium. The toxin interacts with two types of membrane the receptors which belong to different families of neuronal proteins and have different structures. Calcium-dependent receptor of alpha-latrotoxin is identified as neurexin I alpha and belongs to the family of neurexins. This family is selectively expressed in nerve tissue. The calcium-independent receptor of alpha-latrotoxin belongs to the family of G-protein-coupled receptors and proteins which homologous to it are found in heart, lung, kidney and spleen tissues. As a result of alpha-latrotoxin interaction with membrane receptor in the calcium medium the toxin forms the ionic channels in plasmalemma and enhances its calcium permeability. The effects of alpha-latrotoxin on exocytosis in the calcium and calcium-free media and question concerning coupling of channel-forming and secretogenic properties of alpha-latrotoxin are discussed.     

Selective potentiation of drug cytotoxicity by NSAID in human glioma cells: the role of COX-1 and MRP.

Here, we report that nonsteroidal anti-inflammatory drugs (NSAID) enhance the cytotoxic effects of doxorubicin and vincristine in T98G human malignant glioma cells. The cytotoxicity of BCNU, cisplatin, VM26, camptothecin, and cytarabine is unaffected by NSAID. No free radical formation is induced by doxorubicin or vincristine in the absence or presence of NSAID. Doxorubicin and vincristine cytotoxicity in the absence or presence of NSAID are unaffected by free radical scavengers. Functional inhibitors of phospholipase A2 (PLA2), such as dexamethasone and quinacrine, do not mimick the effects of NSAID. T98G cells, but not LN-18, LN-229, LN-308, or A172 glioma cells, express cyclooxygenase (COX-1) and NSAID do not modulate drug cytotoxicity in the other cell lines, except T98G. Thus, augmentation of doxorubicin and vincristine cytotoxicity by NSAID correlates with COX-1 expression. However, ectopic expression of COX-1 in LN-229 cells does not induce the phenotype of T98G cells, indicating that COX-1 inhibition does not mediate the effects of NSAID on drug cytotoxicity. In contrast, a multidrug resistance (MDR) phenotype due to expression of the multidrug resistance-associated protein (MRP) is most prominent in T98G cells and is amenable to modulation by indomethacin, suggesting that inhibition of MRP is at least in partly responsible for the potentiation of doxorubicin and vincristine cytotoxicity by NSAID.