Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes.

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.

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.

Lipid vesicle-cell interactions. I. Hemagglutination and hemolysis.

The interaction of lipid vesicles (liposomes) of several different compositions with erythrocytes has been investigated. Lecithin liposomes, rendered positively charged with stearylamine, exhibit potent hemagglutination activity in media containing low concentrations of electrolytes. The hemagglutination titer is found to be a linear function of the zeta potential of the lipid vesicles. Hemagglutination is reduced when the surface potential of the cells is made more positive by pH adjustment or enzyme treatment. Similarly, hemagglutination is reduced by increasing concentrations of electrolytes. Hemagglutination is examined theoretically and is shown to be consistent with vesicle-cell interactions that are due to only electrostatic forces. Vesicles containing lysolecithin in addition to lecithin and stearylamine cause lysis of erythrocytes, provided the lipids of the vesicles are above the crystal-liquid crystal phase transition temperature. In addition, hemolysis requires close juxtaposition of the vesicle to the cell membrane; vesicles precoated with antibodies exhibit severely diminished hemolytic activities, only a small fraction of which can be attributed to a reduction in hemagglutination titer. Evidence is presented indicating that a single vesicle is sufficient to lyse one cell. With regard to hemagglutination and hemolysis, lipid vesicles of simple composition mimic paramyxoviruses such as Sendai virus.

A differential scanning calorimetry study of the interaction of alpha-tocopherol with mixtures of phospholipids.

When alpha-tocopherol was included in multibilayer vesicles of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine it induced a broadening of the main transition and a displacement of this transition to lower temperatures, as seen by differential scanning calorimetry. This effect was quantitatively more important in the samples of distearoylphosphatidylcholine than in those of the other phosphatidylcholines. Alpha-Tocopherol when present in equimolar mixtures of dimyristoylphosphatidylcholine and diastearoylphosphatidylcholine, which show monotectic behaviour, preferentially partitions in the most fluid phase. The effect of alpha-tocopherol on the phase transition of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine is qualitatively different of that observed on phosphatidylcholines, and several peaks are observed in the calorimetric profile, probably indicating the formation of separated phases with different contents in alpha-tocopherol. The effect was more apparent in dipalmitoylphosphatidylethanolamine than in dilauroylphosphatidylethanolamine. The inclusion of alpha-tocopherol in equimolar mixtures of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylcholine, which show cocrystallization, only produced a broadening of the phase transition and a shift to lower temperatures. However, in the case of equimolar mixtures of dipalmitoylphosphatidylcholine which also show cocrystallization, the effect was to cause lateral phase separation with the formation of different mixtures of phospholipids and alpha-tocopherol. Alpha-Tocopherol was also included in equimolar mixtures of phosphatidylethanolamine and phosphatidylcholine showing monotectic behaviour, and in this case alpha-tocopherol preferentially partitioned in the most fluid phase, independently of whether this was composed mainly of phosphatidylcholine or of phosphatidylethanolamine.

Enzyme therapy. VI: Comparative in vivo fates and effects on lysosomal integrity of enzyme entrapped in negatively and positively charged liposomes.

Entrapment of enzyme in liposomes, biodegradable lipid vesicles, offers an intriguing strategy for the intracellular delivery of these macromolecules to the lysosomal apparatus for enzyme replacement endeavors in selected lysosomal storage diseases. Therefore, the in vivo tissue and subcellular fate and effect on the subcellular distribution of endogenous lysosomal hydrolases was determined following intravenous administration of beta-glucuronidase entrapped in positively and negatively charged liposomes into C3H/HeJ beta-glucuronidase-deficient mice. Enzyme entrapped in negatively charged liposomes was rapidly cleared from the circulation (t1/2 approximately 4 min); maximal tissue recovery, 75% of dose, was detedtec in the liver at 1 h, was maintained fro 48 h and then gradually declined to non-detectable levels by 8 days. A similar circulatory clearance and reciprocal hepatic uptake was observed fro positively charged liposomes; however, the beta-glucuronidase was retained in murine liver for 11 days. Significant activity, 15% of dose, was found in the kidneys up to 1 and 4 days post-injection of positively and negatively charged liposomes, respectively. No activity was recovered in neural or other visceral tissues except in spleen and lungs (less than 5% of the dose). Exogenous beta-glucuronidase activity administered in negatively charged liposomes was primarily localized in the lysosomally-enriched hepatic subcellular fraction, compared to the predominantly soluble localization of exogenous activity entrapped in positively charged liposomes. Administration of negatively charged liposomes caused no detectable change in the subcellular localization of several endogenous lysosomal hydrolase activities compared to their distribution in untreated mice. In contrast, a marked but temporary translocation of these hydrolase activities into the soluble fraction was observed following the administration of positively charged liposomes, identifying possible deleterious effects on cellular physiology.

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids.

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness. These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.

Induction of apoptosis in macrophages by cationic liposomes.

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.

[Calcium, modulator of the expression of gonadoliberin at the intracellular level]. / Le calcium, modulateur de l'expression du message de la gonadoliberine au niveau intracellulaire.

GnRH has been entrapped in liposomes. Chromatographic studies and enzymatic peptidase treatments, show the efficiency of the encapsulation. A purification method on G75 Sephadex of the entrapped GnRH is described. This method prevents any dilution of the liposome fraction. A free GnRH contamination, lower than 0.4 per cent, has been observed. Superfused hypophyses respond to the message of the internalized GnRH only when calcium is present in the extracellular medium. The intensity of the answer depends on the duration of the entrapped GnRH infusion. The decrease observed in the response intensity after a long stay of the GnRH in the cytoplasm allows us to say that GnRH controls its own expression: The binding of GnRH to the membrane receptor during the early phase induces a calcium uptake necessary to the expression of the internalized GnRH, this being the late phase in LH release. A too low calcium concentration does not allow GnRH expression. As a consequence, GnRH is enzymatically degradated by the cytoplasmic peptidases. The LH release during the late phase is the result of a combined action of calcium and cytoplasmic peptidases. To support this idea we show: 1- that an extracellular calcium concentration around 0.5 or 0.6 mM is the best condition for the expression of the internalized GnRH. 2- that a GnRH agonist (D-Ala6-GnRH) known to be peptidase resistent induces a higher LH release in our experimental conditions.

Lens junctions are communicating junctions.

Lens fibers are electrically coupled with each other and directly exchange dyes and metabolites. In most cells, this form of communication is mediated by gap junctions. Lens fibers lack typical gap junctions. The lens junctions, although morphologically similar to gap junctions, differ from them structurally, chemically and immunologically. Nevertheless, recent evidence suggests that indeed lens junctions are communicating junctions. The lens junction protein, MIP26, displays structural characteristics similar to other channel proteins. Once incorporated into liposomes it forms channels permeable to molecules as heavy as 1.5 kDa. Like other communicating junctions, lens junctions assume crystalline arrays and uncouple with Ca++. The liposome incorporated channels close with Ca++ and H+ in the presence of calmodulin (CaM). Partial loss of gating competency occurs after proteolytic cleavage of the C-terminal arm of MIP26. The need for a unique type of communicating junction in lens is unclear. A possibility is that this tissue has some special cell-to-cell transport requirements, in terms of size and/or charge of permeants, not shared by coupled cells of other tissues.

Measurements of viscosity of synthetic erythrocyte suspensions.

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.

Fluidity and osmotic sensitivity changes of phospholipase A2-treated liposomes.

Membrane fluidity and osmotic sensitivity were examined in DPPC liposomes treated with phospholipase A2 (PL.A2) in the presence of Ca2+ or Mg2+. The amount of liposome phospholipid hydrolyzed differed with the two ions. Embedded DPH, a rod-like fluorescent probe, was employed in the determination of membrane fluidity. Membrane fluidity decreased according to the degree of phospholipid hydrolization in liposomes by PL.A2. The reciprocal value of absorption at 450 nm was measured as the index of osmotic sensitivity of liposomes. Intact sonicated liposomes showed osmotic insensitivity. PL.A2-treated liposomes in which about 40% of total phospholipid was hydrolyzed showed osmotic sensitivity. No change in the membrane fluidity was obtained when PL.A2-treated liposomes were exposed to hypertonic or hypotonic solution. These results suggested that the motion of the acyl-chain of phospholipids and free fatty acids was resisted in PL.A2-treated liposomes. The resistance may be due to a phase separation between phospholipids and free fatty acids. The pore for water permeation might be induced in the border between phase-separated domains in PL.A2-treated liposomes.

Purification and biochemical characterization of pulmonary surfactant protein A of horses.

OBJECTIVE: To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses. ANIMALS: 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease. PROCEDURE: Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins. RESULTS: Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies. CONCLUSION: Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses. CLINICAL RELEVANCE: Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.

[The function of glutamate receptors in the membrane vesicules, proteoliposomes and hybrid cells of the neuroblastoma N18Tg2a]. / Funktsii glutamatnykh retseptorov v membrannykh vezikulakh, proteoliposomakh i gibridnykh kletkakh neiroblastomy N18Tg2a.

The transport and selective functions of the glutamate-binding proteins of the rat brain cortex synaptic membranes, were studied. The data on kinetics of absorption of the ions 22Na+, 86Rb+ and 45Ca++ by the membrane vesicles and liposomes containing receptor proteins, are presented. The specific features of the c. n. s. glutamate receptors functioning in the hybrid cells of the neuroblastoma N18Tg2a, are revealed. A selective activation of transport of the 22Na+ ions was found in this kind of model systems in presence of physiological concentrations of L-glutamate. The modelling of the glutamate receptors function depended on composition of lipids, presence of the endogenous peptide agent inhibiting binding of the H3-L-glutamate, and on the degree of the neuroblastoma differentiation. Monoclonal antibodies obtained for the receptor's recognizing areas blocked the functions of the glutamate-binding proteins in all the model systems.

Ca++-induced fusion of proteoliposomes: dependence on transmembrane osmotic gradient.

The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 A lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 mumM have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.

Interactions of liposomes with mammalian cells.

In this review we have attempted to highlight each of the major areas of interest in liposome-cell interactions: the purely physical chemical, the cell biological, and the medical. Liposomes can be generated in a number of ways and are classified as small unilamellar, large unilamellar, and multilamellar vesicles. Although liposomes are easy to prepare, it is important to consider the effects of impurities, and also the possible changes in liposome properties with time (particularly at or below the phase transition temperature). Intelligent application of liposomes to cell biological and clinical problems requires an understanding of their mechanisms of interaction with cells. The mechanisms thus far delineated, largely by studies in vitro, are fusion, endocytosis, lipid transfer, and stable adsorption. In practice, demonstrating the occurrence of a given mechanism in an actual system is difficult because these are not mutually exclusive. Cell type, conditions of incubation, and liposome properties (charge, fluidity, size) are important in determining mechanism and appear to organize the literature effectively. However, this may be an oversimplification resulting from the sketchiness of current information. Liposomes have been used in cell biology to alter the phospholipid and cholesterol composition of cells, to bypass the membrane permeability barrier to normally impermeant solutes, and to promote cell-cell fusion. Perhaps the most fruitful of these applications has been the alteration of cholesterol, which can result in changes in cell permeability and morphology. On the other hand, delivery into cells of liposome-entrapped, water-soluble materials has not yet proved an effective tool in cell biology; delivery, and consequent physiological changes, have been demonstrated, but generally to answer questions about liposome-cell interactions, not to answer questions about the cells. Much of the current interest in liposomes derives from their potential applications in vivo. Liposomes are envisioned as pharmacological capsules for delivery of therapeutic agents in treatment of such conditions as diabetes, enzyme deficiencies, heavy metal poisoning, and neoplasms. Although much of the literature to date has been concerned with the end applications, it seems clear that a more systematic approach to the pharmacokinetics of liposomes will be necessary. In particular, such aspects as their leakage rates and their ability to cross cell and anatomical barriers require further study. Targeting of liposomes to particular cells or tissues will be essential for many applications. Finally, it must be remembered that all of these in vivo applications of liposomes are future tense; as with other technologies, passage from demonstration of the phenomenon to practical application is likely to be arduous.

Enkephalin-cell interactions.

Enkephalin analogs for multivalent ligand systems, bivalent enkephalins, multivalent enkephalins on polymers, multivalent ligands on vesicles, simultaneous activation of two different receptor systems, and cell interactions of enkephalin/polypeptide conjugates are described. Multivalent ligand systems can trigger receptor-receptor interactions and are considered to possess interesting possibilities in terms of enhanced potency, reduction of side effects, and a new biological activity.