Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes.

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.

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.

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.

Transcytotic vesicle fusion is reduced in cholestatic rats: redistribution of phospholipids in the canalicular membrane.

Cholestasis, which affects phospholipid trafficking, therefore would be expected to alter canalicular membrane phospholipid composition and fluidity, as well as fatty acid composition of membrane phospholipid. These alterations may affect transcytotic vesicle fusion and would be expected to cause variety of cholestatic phenomena. The aim of this study was to determine the effect of cholestasis on transcytotic vesicle fusion. Sprague-Dawley rats with extrahepatic and intrahepatic cholestasis were prepared by bile duct ligation (6 hr or three days) and phalloidin infusion (0.4 mg/kg body weight), respectively. Liposomes of phosphatidylserine/phosphatidylcholine were labeled with octadecyl rhodamine B chloride. Fusion of liposomes to canalicular membrane vesicle preparations from cholestatic and control rats was induced by the addition of calcium. The degree of fusion was evaluated by measuring the increase in rhodamine fluorescence. Membrane phospholipid content also was analyzed. Rates of liposomal fusion to membranes from cholestatic rats were decreased compared to controls. The saturated/unsaturated and saturated/polyunsaturated fatty acid ratios were increased in membrane phosphatidylcholine and decreased in membrane sphingomyelin from cholestatic rats. Cholesterol/phospholipid ratios were increased. Thus, in the presence of cholestasis, a redistribution of phospholipid species within canalicular membranes is associated with decreased transcytotic vesicle fusion. Cholestasis likely decreases membrane fluidity. The regulation of phospholipid species within hepatocellular membranes may play an important role in intrahepatic lipid transport.

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells.

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.

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.

Poly(ethylene glycol) (PEG)-mediated fusion between pure lipid bilayers: a mechanism in common with viral fusion and secretory vesicle release?

Membrane fusion is fundamental to the life of eukaryotic cells. Cellular trafficking and compartmentalization, import of food stuffs and export of waste, inter-cellular communication, sexual reproduction, and cell division are all dependent on this basic process. Yet, little is known about the molecular mechanism(s) by which fusion occurs. It is known that fusing membranes must somehow be docked and brought into close contact. Specific proteins, many of which have been identified within the past decade, accomplish this. An electrical connection or 'fusion pore' is established between compartments surrounded by the fusing membranes. Three primary views of the mechanism of pore formation during secretory and viral fusion have been proposed within the past decade. In one view, a protein ring forms an initial transient connection that expands slowly by recruiting lipid so as to form a lipidic junction. In another view, the initial fusion pore consists of a protein-lipid complex that transforms slowly until the fusion proteins dissociate from the complex to form an irreversible lipidic pore. In a third view, the initial pore is a transient lipid pore that fluctuates between open and closed states before either expanding irreversibly or closing. Recent work has helped define the mechanism by which poly(ethylene glycol) (PEG) mediates fusion of highly curved model membranes composed only of synthetic phospholipids. PEG is a highly hydrated polymer that can bring vesicle membranes to near molecular contact by making water between them thermodynamically unfavourable. Disrupted packing in the contacting monolayers of these vesicle membranes is necessary to induce fusion. The time course and sequence of molecular events of the ensuing fusion process have also been defined. This sequence of events involves the formation of an initial, transient intermediate in which outer leaflet lipids have mixed and small transient pores join fusing compartments ('stalk'). The transient intermediate transforms in 1-3 min to a fusion-committed, second intermediate ('septum') that then 'pops' to form the fusion pore. Inner leaflet mixing, which is shown to be distinct from outer leaflet mixing, accompanies contents mixing that marks formation of the fusion pore. Both the sequence of events and the activation energies of these events correspond well to those observed in viral membrane fusion and secretory granule fusion. These results strongly support the contention that both viral and secretory fusion events occur by lipid molecule rearrangements that can be studied and defined through the use of PEG-mediated vesicle fusion as a model system. A possible mechanism by which fusion proteins might mediate this lipidic process is described.

Candidacidal activity of shortened synthetic analogs of amoebapores and NK-lysin.

Natural antimicrobial peptides and synthetic analogs thereof have emerged as compounds with potentially significant therapeutical application against human pathogens. Amoebapores are 77-residue peptides with cytolytic and antibacterial activity considered to act by forming ion channels in cytoplasmic membranes of the victim cells. A functionally and structurally similar peptide named NK-lysin exists in mammalian lymphocytes. Several synthetic analogs of amoebapores and NK-lysin, which are substantially reduced in size compared to the parent molecules, were tested for their ability to inhibit the growth of and to kill Candida albicans. Some of the peptides displayed potent activity against a clinical isolate as well as against defined culture strains. Among the most active peptides found are some shortened substitution analogs of amoebapore C and a cationic core region of NK-lysin. As these peptides are also highly active against Gram-positive and Gram-negative bacteria but are of low cytotoxicity towards a human keratinocyte cell line they may provide promising templates for the design of broad-spectrum peptide antibiotics.

Liposomes can function as targets for natural killer cytotoxic factor but not for tumor necrosis factor.

NK cells exert their lytic action through the release of NK cytotoxic factors (NKCF) after stimulation by the bound target cell. NKCF may be related to granule-derived perforin/cytolysin on one hand and to the pleiotropic cytokine TNF on the other hand. In the present study, we show that NKCF can also lyse artificial lipid vesicles, as had been reported previously for cytotoxic granules and cytolysin. The lysis of large unilamellar vesicles was monitored by measuring the release of the encapsulated fluorescent dye carboxyfluorescein. NKCF-induced lysis was only observed with liposomes composed of a complex mixture of lipids including acidic phospholipids. No lysis could be demonstrated if the liposomes contained phosphatidylcholine as the only phospholipid, suggesting some kind of lipid specificity for the action of NKCF. A remarkable finding was that neither recombinant nor natural TNF were able to lyse large unilamellar vesicles, irrespective of their lipid composition, indicating different ways of interaction of NKCF and TNF with artificial (and presumably also biological) membranes.

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.

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.

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.

Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies.

Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K+ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at -80 degrees C and room temperature, respectively. The liposomes were assayed for the SR K+ channel using the patch clamp technique. Channel density was modulated by varying protein:lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range +/- 70 mV and has a conductance of 178.4 +/- 4.4 pS (mean +/- SE, n = 37) in 200 mM KCl. The channel has a selectivity sequence of K+ greater than NH4+ greater than Rb+ greater than Na+ and K+ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.

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.

[Electrical stability of mitochondrial membranes: the role of thyroid hormones and the fat component of the diet]. / Elektricheskaia stabil'nost' membran mitokhondriii: rol' tireoidnykh gormonov i zhirovogo komponenta diety.

Electric stability of the membranes of the mitochondria and liposomes formed from mitochondrial lipids was studied. The mitochondria were isolated from the liver of euthyroid or hyperthyroid rats kept on the diets with varying degree of food fat unsaturation. In the first group animals, butter was used as a fatty component of the diet whereas the second group animals received sunflower oil. The electric stability of the membranes of the mitochondria and respective liposomes appeared lower in the first group animals as compared with those in the second group animals. Hyperthyrosis was accompanied by the increased electrical stability of mitochondrial lipids in both the groups. At the same time the liposomal membranes were similar as regards the electric stability, whereas the electric stability of the mitochondrial membranes in the first group hyperthyroid and euthyroid rats was lower than in the organelles of the second group animals. It is thus assumed that the electric stability of the mitochondria is determined not only by the chemical composition of lipids but also by other factors.

[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.

The current-voltage relationships of liposomes and mitochondria.

Current-voltage relationships were determined for various membrane systems. We show that phospholipid and mitochondrial membranes exhibit linear relations between H+ flux and pH gradients. These membranes, however, exhibited non-linear relationships when the applied voltage was a membrane potential. The current-voltage relationship approximated to an exponential function. This relationship was found to be linearized when the membranes were treated with an electrogenic proton ionophore. The incorporation of cytochrome c oxidase (EC 1.9.3.1) was found to have no effect on the current-voltage characteristics of the phospholipid membranes. When a membrane potential of more than 140 mV was imposed across vesicular and mitochondrial membranes, they exhibited reversible di-electric breakdown. This phenomenon was correlated with the requirement of a permeant ion for the experimental demonstration of proton translocation by so-called 'proton pumps'.

[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.

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.