Control of electric field induced cell membrane permeabilization by membrane order

Cells can be made temporarily permeable if pulsed by high-intensity short-duration electric fields. The molecular mechanisms underlying this electropermeabilization are still unknown. The kinetic events may be described by four successive steps: induction, expansion, stabilization, and resealing. On one hand, cell electropermeabilization is detected only under more stringent conditions when cells have been treated by ethanol. On the other hand, lysolecithin is observed to facilitate cell electropermeabilization. More precisely, these molecules that modify membrane order, when used in concentrations compatible with cell viability, are shown to affect only the expansion and resealing steps. Electropermeabilization is inducing a transition in the membrane organization. Membrane order is modulating the energy barrier needed to evoke this membrane transition which occurs when cells are submitted to a field larger than a characteristic threshold (expansion step). Less order would increase the magnitude of this energy barrier; more order would decrease it.     

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.     

Reversibly permeable hepatoma cells in culture

A brief treatment of H35 hepatoma cells with lysolecithin resulted in a cell population which is permeable to low-molecular weight charged molecules that cannot normally cross the plasma membrane. These include deoxynucleotide and nucleotide triphosphates, folyl and methotrexate polyglutamates, and trypan blue. As a result dTTP can be incorporated into the DNA of the permeable cells, providing the required nucleotides and deoxynucleotides are added to the medium. This result, combined with only a slight observed loss (20–25%) in total cell protein, lactate dehydrogenase (EC 1.1.1.27) activity and tyrosine aminotransferase (EC 2.6.1.5) activity, demonstrated that permeation of the cells does not extensively disrupt membrane integrity. Further support for this view comes from the fact that the permeable cells could seal when placed in enriched medium. The process of sealing was inhibited by cycloheximide and tunicamycin. The sealed cells, whose surfaces appeared identical to those of untreated cells by scanning electron microscopy, were fully capable of cell division when exposed to serum. Values for several other parameters, including dexamethasone-dependent tyrosine aminotransferase induction, thymidine incorporation into DNA, leucine incorporation into protein and folate coenzyme transport, supported the conclusion that sealed cells and untreated H35 cells have identical properties. Based on the characteristics of the permeable and sealed H35 cells, a discussion of the experimental potential of these preparations for studying macromolecular synthesis, investigating enzymes in situ and depleting cells of folate coenzymes is presented.     

A procedure to introduce protein molecules into living mammalian cells

Although several methods are now available by which to introduce macromolecules into cultured living mammalian cells, each has limitations on its adoption as a general means, for a variety of purposes. We describe here a simple procedure to introduce protein molecules into various living mammalian cells. This procedure is based upon the finding that mammalian cells, after exposure to a low concentration of a phospholipid (L-alpha-lysophosphatidylcholine) in the presence of high (hypertonic) concentrations of glycerol became permeable to protein molecules and that a significant portion of the exposed cells regain their viability following incubation in the appropriate growth medium. We have demonstrated that diphtheria toxin (A fragment), horseradish peroxidase and antibodies against SV40 T-antigens are incorporated into living mouse erythroleukemia (Friend) cells, baby hamster kidney (BHK) cells and mouse fibroblasts (C3H), respectively. The volume introduced into a single cell (mouse Friend cells) is approx. 3 X 10(-15) liter, which is comparable to those with other systems. Parameters affecting permeability to protein molecules and viability of the treated cells were also investigated with these and other cell lines.     

Human high denisty apolipoprotein A-I-lysolecithin-lecithin and sphingomyelin complexes. A method for high yield recombinations to lipoprotein complexes of reproducible stoichiometry.

High denisty apolipoprotein A-1 (apoLp A-I) has been prepared in a chromatographically and immunochemically homogeneous form. This apoprotein forms trimeric and tetrameric aggregates in aqueous solutions at higher concentrations. ApoLp A-I has been recombined in almost quantitative yield in the presence of lysolecithin with phosphatidylcholine and sphingomyelin to particles of reproducible stoichiometry. Lysolecithin is not required for the interactions of lecithin and sphingomyelin with the apoprotein A-I or for the stability of these complexes. Dialysis removes most of the lysolecithin without the loss of lecithin and sphingomyelin. ApoLp A-I-lecithin particles have a molecular weight of 200 000 and contain 50 molecules lecithin and 25 of lysolecithin. ApoLp A-I-sphingomyelin complexes contain 50 sphingomyelin and 13 lysolecithin molecules. The former particles show up as discs of 100 A diameter, and the latter particles are 250 A in diameter. Their thickness was estimated as 25 A in the apoLp A-I lecithin and 60 A in the apoLp A-I-sphingomyelin particles. ApoLp A-I and lysolecithin form complexes whose densities depend on the lysolecithin concentration. Lysolecithin enhances the binding of phosphatidylcholine to apoLP A-I, yielding lipoprotein complexes with decreasing density. The yield of apoLp A-I-sphingomyelin-lysolecithin complexes is proportional to the lysolecithin concentration. The ratio of apoLp A-I to sphingomyelin in all these complexes remains constant.     

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.     

Lipid vesicle-cell interactions. II. Induction of cell fusion

The ability of lipid vesicles of simple composition (lecithin, lysolecithin, and stearylamine) to induce cells of various types to fuse has been investigated. One in every three or four cells in monolayer cultures can be induced to fuse with a vesicle dose of about 100 per cell. At such dosages and for exposures of 15 min to 1 h, vesicles have essentially no effect on cell viability. Under anaerobic conditions, these cells lyse rather than fuse. Avian erythrocytes are readily fused with lipid vesicles in the presence of dextran. Fusion indices increase linearly with the zeta potential of the vesicles (increasing stearylamine content), indicating that contact between vesicle and cell membrane is required. Fusion indices increase sublinearly with increasing lysolecithin content. Divalent cations increase fusion indices at high vesicle doses. The data presented are consistent with the hypothesis that cell fusion occurs via simultaneous fusion of a vesicle with two adhering cell membranes.     

Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).     

A test of granulocyte membrane integrity and phagocytic function.

An assay of granulocyte viability has been developed which yields information about rwo important cell parameters, cell membrane integrity and phagocytic activity. The assay is based on the fact that only live cells can accumulate fluorescein, which is enzymatically split from the nonfluorescent substrate fluorescein diacetate. Dead cells, on the other hand, become permeable to the fluorescent red dye ethidium bromide. When cells are exposed first to opsonized zymosan particles, which they can phagocytize, then to a combination of these fluorescent dyes, one can distinguish microscopically between dead cells with fluorescent red nuclei, live cells which fluoresce green, and live cells with phagocytic function which are swollen with the pink zymosan particles in a green fluorescing cytoplasm. This assay takes 20--30 min and can be used to distinguish different degrees of cellular damage after cryopreservation.     

Induction of homokaryocyte, heterokaryocyte and hybrid formation by lysolecithin

Lysolecithin has been utilized to induce homo- and heterokaryocyte formation in CV-1, F5-1 and WI-38 cells and hybrid formation in 1R and mKSBu100 cells cultured in vitro. A new fusion technique utilizing solutions of albumin or delipidized serum as vehicles for lysolecithin has been introduced. Optimal concentrations of lysolecithin for inducing cell fusion and reducing cell damage for various cell lines have been evaluated. Lysolecithin produced 15–25% homokaryocytes and about 5–7% heterokaryocytes. Two different mutant cell lines fused in the presence of lysolecithin produced hybrids which survived in selective medium.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

We study exocytosis in the planar isolated cortex of the egg of the sea urchin Lytechinus pictus. Solutions bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause secretion in vitro. We add the amphipathic compound digitonin at 12 to 15 microM concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.     

Intracellular polycationic molecules cause reversible swelling of the rough endoplasmic reticulum

The microinjection of polycationic but not anionic molecules causes swelling of the rough endoplasmic reticulum (RER) in salivary gland cells of a fly larva. Ca-EGTA buffers, lanthanum chloride, lysozyme, bovine serum albumin, cationic and anionic ferritin were microinjected into salivary gland cells and their effects observed by light and electron microscopy. Immediately after the microinjection of polycationic molecules, the cytoplasm changed from transparent to opaque as the RER became swollen. Binding of polycationic molecules to the RER may cause the membrane to become permeable to some solute and swell due to osmotic forces.     

Differential scanning calorimetry on mixtures of lecithin,lysolecithin and cholesterol

The effect of increasing concentrations of lysolecithin (1-palmitoyl-sn-glycerol-3-phosphorylcholine) on the gel → liquid crystal thermal transition of lecithin (1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine) in the aqueous phase was studied by differential scanning calorimetry (DSC). Lysolecithin showed an endothermic transition at 3.4°C whereas the transition of the lecithin occurred at 42°C. No phase separation could be observed calorimetrically at lysolecithin concentrations up to 60 mol%. Freeze etch electron microscopy showed that mixtures containing as much as 50 mol% lysolecithin exist in a lamellar phase. The lysolecithin was found to cause an initial slight increase in the enthalpy of transition followed by a gradual decrease. The enthalpy increased again at very high lysolecithin concentrations. The lysolecithin also caused a non-linear decrease in the temperature at which the lecithin transition took place.Cholesterol was found to decrease the enthalpy of transition of the lysolecithin, eliminating it at a concentration of 50 mol%. Cholesterol caused an increase in the temperature at which the lysolecithin transition took place.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Lysolecithin-induced fusion of fibroblasts

Summary Lysolecithin has been used to induce cell fusion between two metabolically deficient mouse fibroblast lines, A₉ and B₈₂. Attempted fusion in suspension led to excessive cell clumping and complete loss of viability. Addition of lysolecithin solutions to confluent monolyers caused extensive detachment of cells from glass or plastic surfaces. At higher levels of lysolecithin few cells survived. When the conditions were controlled (50 to 250 μg per ml for up to 20 min), extensive polykaryocyte formation was observed. In the presence of selective medium (HAT) colonies of hybrid cells grew and a series of cell strains were isolated. The presence of inosinic acid pyrophosphorylase (absent in A₉ cells) was demonstrated in the hybrid cells which were shown to have almost double the cell volume of the parent A₉ and B₈₂ cells. Unlike the parent cell lines, the hybrid cells grew well in the presence of HAT both as monolayers and in suspension.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

Summary We study exocytosis in the planar isolated cortex of the egg of the sea urchinLytechinus pictus. Solutins bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause seretion in vitro. We add the amphipathic compound digitonin at 12 to 15 M concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Restricted Diffusion in Cellular Media: (1+1)-Dimensional Model

We consider the diffusion of molecules in a one-dimensional medium consisting of a large number of cells separated from the extra-cellular space by permeable membranes. The extra-cellular space is completely connected and allows unrestricted diffusion of the molecules. Furthermore, the molecules can diffuse within a given cell, i.e., the intra-cellular space; however, direct diffusion from one cell to another cell cannot occur. There is a movement of molecules across the permeable membranes between the intra- and extra-cellular spaces. Molecules from one cell can cross the permeable membrane into the extra-cellular space, then diffuse through the extra-cellular space, and eventually enter the intra-cellular space of a second cell. Here, we develop a simple set of model equations to describe this phenomenon and obtain the solutions using an eigenfunction expansion. We show that the solutions obtained using this method are particularly convenient for interpreting data from experiments that use techniques from nuclear magnetic resonance imaging.     

Effect of glycerol on plasmid transfer in genetically competent Haemophilus influenzae

Summary The small plasmid pAT4 transformed at characteristically low frequencies those competent Haemophilus influenzae Rd strains that had no DNA homology with this plasmid. Transformation was increased up to 100 times, however, when the recipient cells were exposed to 30% glycerol before plating for transformants. Expression of plasmid resistance markers was then immediate. Ultraviolet irradiation experiments indicated that this large increase was due to release by the glycerol of double-stranded plasmid molecules, presumably from transformasomes. Several other plasmids exhibited the same phenomenon. Dimethylsulfoxide also stimulated plasmid transformation but lysolecithin and high concentrations of NaCl or glucose were ineffective. Glycerol did not increase the efficiency of transformation by either chromosomal DNA or linearized plasmid DNA.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane: III. Discrimination between the causal rôle of split products and of lecithin removal

Summary Cleavage of 55% of the lecithin in intact human erythrocytes by phospholipase A₂ (bee venom) markedly inhibits the mediated transport ofl-lactate (via the monocarboxylate carrier) and ofl-arabinose (via the monosaccharide carrier), while the major anion exchange system (probed by oxalate) and diffusion via the lipid domain (probed by erythritol) remain essentially unaltered. The causal role of the split products, unsaturated fatty acids and saturated lysolecithin, and of lecithin removal were now studied by sequential extraction of split products with serum albumin and by their controlled insertion into normal membranes. Careful choice of the albumin-to-cell ratio allowed the extraction of more than 95% of the fatty acids and up to 80% of the lysolecithin without hemolysis.Extraction of fatty acids abolished inhibition of lactate and arabinose transfer, but induced inhibition of anion exchange and translipid permeation. Subsequent extraction of lysolecithin produced no further effects except on lactate transfer, which was inhibited.Exogenous oleic and linoleic acid, at intramembrane concentrations equal to those produced by phospholipase A₂, inhibit lactate and arabinose transfer, while accelerating oxalate and erythritol movements, in agreement with effects of endogenous fatty acids. Exogenous lysolecithin inhibits all mediated transfer processes but does not alter translipid permeation. This pattern differs from that obtained for endogenous lysolecithin.The action of exogenous lysolecithin can be suppressed by loading of the cells with cholesterol. Insertion of exogenous lysolecithin into cells depleted of endogenous lysolecithin does not restore the functional state before depletion, indicating that exogenous and endogenous lysolecithin may act differently.Modification of membrane phospholipids by cleavage with phospholipases has been used by many investigators to study the relevance of lipids for protein-related functions of biomembranes. In many instances pronounced effects could be demonstrated. With the exception, however, of electrical characteristics of neurons [21] and axons [39], the properties investigated only comprised the binding of toxins, drugs [4, 28], transmitters [1], and hormones [2, 48] to their receptors, or enzymatic reactions [5, 10, 11, 13, 36, 37, 43].In previous investigations [49, 50] of this series we have analyzed the effect of enzymatic cleavage of exofacial membrane phospholipids (phosphatidylcholine, sphingomyelin) on simple translipid, and on facilitated, protein-mediated diffusion processes across the human erythrocyte membrane. Rates of nonelectrolyte movements via the lipid domain and of mediated exchange of inorganic anions remained essentially unaltered after hydrolysis of up to 60% of the phosphatidylcholine, corresponding to about 18% of the membrane phospholipids or 36% of those in the outer leaf of the lipid bilayer. In contrast, the movements ofl-arabinose, catalyzed by the monosaccharide carrier system, and ofl-lactate, transported by a specific monocarboxylate carrier, were markedly inhibited by phospholipid cleavage. In similar studies, inhibition of the active extrusion of Na⁺ has recently been demonstrated in human erythrocytes treated with phospholipase A₂ [14]. These results obtained on erythrocytes provided first evidence for effects of phospholipid cleavage on solute translocation across biomembranes in intact cells.Inhibitory effects of phospholipid cleavage can in principle be due either to the production of the split products, lysolecithin and fatty acid, which remain bound to the membrane, or to the disappearance of a particular phospholipid. In order to distinguish between these possible mechanisms, two procedures can be used. First, the split products of lecithin, although tightly bound to the membrane core, can be removed by treatment with serum albumin. Second, split products can be introduced into the membrane of normal cells. If the former procedure abolishes and the latter one mimics the effects of phospholipase A₂ treatment, split products are likely to be responsible for the effects of phospholipase A₂. Otherwise, the disappearance of a native phospholipid has to be considered.Testing the removal of split products is easily accomplished in isolated membranes [10, 11, 13, 37, 43], but has met problems in intact erythrocytes, which lysed after extraction of part of the split products in earlier studies [17]. Comparisons between the actions of exogenous and endogenous fatty acid and lysolecithin, on the other hand, were mostly qualitative as yet, since effects were related to bulk concentrations of the exogenously added substances and not to thosewithin the membrane.The following attempt to further clarify the effects of phospholipase A₂ treatment on erythrocytes is based on a stepwise, controlled extraction of endogenous split products and a quantitative evaluation of the action of exogenous split products. From the results it will become evident that transport processes in the same membrane may differ markedly with respect to the mechanisms by which cleavage of phosphatidylcholine exerts its effects.