Ionophore-mediated loading of Ca2+ into large unilamellar vesicles in response to transmembrane pH gradients.

The Ca2+ translocating properties of the carboxylic ionophores A23187, ionomycin and lasolocid A (X537A) have been investigated by employing large unilamellar vesicles that exhibit a pH gradient (acidic interior). An analysis of Ca2+ uptake at equilibrium reveals that Ca2+ accumulation is an electroneutral process, whereby one Ca2+ ion is transported in for every two H+ ions transported out. A kinetic analysis shows that both A23187 and ionomycin transport Ca2+ in the form of a 1:1 cation:ionophore complex, whereas a 1:2 complex is observed for lasolocid A. The specificity of the ionophores for transporting Ca2+ is reflected by the influence of exterior Na+ ions that inhibit Ca2+ uptake for lasolocid A but do not influence ionomycin-mediated uptake.

Imaging of skin epidermis from various origins using confocal laser scanning microscopy.

The ability to image the subsurface morphology of intact three-dimensional matrices such as skin epidermal tissue, using conventional optical microscopy, requires the tissue to undergo an extensive preparative protocol. This protocol includes fixing, embedding and physically sectioning the specimen. The process is slow and subject to sample perturbation, which can result in image misinterpretation. In this study, confocal laser scanning microscopy is used successfully for the imaging of the surface and underlying morphology of unlabelled and fluorescently labelled intact skin epidermal tissue, from various origins (that is human, pig and cultured epidermis). Unlike conventional optical microscopy, the technique requires little or no pretreatment of the tissue prior to imaging. Hence the technique is rapid, and the tissue experiences minimal physical perturbation and/or damage. Confocal images of skin epidermis in the reflected light (unlabelled) and fluorescent modes were obtained. In the reflected-light mode the detailed morphology of the various layers of the intact epidermis (that is the strata corneum, granulosum, spinosum and basale) from a variety of sources are clearly imaged. In the fluorescent mode, environment-specific information is obtained. For example, topical application of the fluorescent probe acridine orange readily allows the confocal imaging of the nuclear material (nuclei and nucleoli), from cells of the different strata of intact epidermis. In the case of the fluorophore Nile red, however, visualisation of the lipid material of the epidermis, including the intercellular lipid of the stratum corneum and the lipid granules of the stratum granulosum, is possible. he reflectance and fluorescence confocal images are sel-consistent and agree with images obtained by conventional, physical sectioning protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and characterization of iron- and barium-loaded liposomes.

Previous work (Veiro and Cullis (1990) Biochim. Biophys. Acta 1025, 109-115) has shown that Ca2+ can be accumulated into large unilamellar vesicles (LUVs) in the presence of a transmembrane pH gradient (inside acidic) and the Ca(2+)-ionophore A23187. Here, the ability of A23187 to mediate the uptake of iron and barium into LUVs has been investigated. It is shown that under appropriate conditions of temperature and A23187 concentration, iron (in the form of Fe2+) can be accumulated into EPC and DSPC/cholesterol (55:45; mol/mol) LUVs with an acidic interior. This uptake is dependent on the internal buffer concentration, with maximum levels of uptake in the range of 300 nmol of cation per mumol lipid. The DSPC-cholesterol LUV systems exhibit superior retention properties compared to the EPC systems. It is demonstrated that Ba2+ can also be loaded by similar methods. It is also shown that the maximally loaded Fe(2+)- and Ba(2+)-containing LUVs exhibit increased densities. This is expressed by enhanced gravimetric properties, as an increased proportion of the loaded LUVs can be pelleted by low speed centrifugation, and by enhanced electron densities, in that the Ba(2+)-loaded systems can be directly visualized employing cryo-electron microscopy.

A novel method for the efficient entrapment of calcium in large unilamellar phospholipid vesicles.

A technique for the efficient entrapment of high concentrations of Ca2+ in large unilamellar phospholipid vesicles (LUVs), using the carboxylic acid antibiotic ionophore A23187 (calcimycin) is demonstrated. It is shown that rapid A23187-mediated entrapment of Ca2+, corresponding to essentially 100% sequestration of the extravesicular cation may be achieved for egg yolk phosphatidylcholine LUVs (100 nm) in the presence of a transmembrane proton gradient (acidic interior). Interior-exterior concentration cation gradients of over 400-fold may be readily achieved, with interior Ca2+ concentrations in excess of 250 mM. It is shown that the extent and efficiency of the A23187-mediated uptake process is affected by the intravesicular buffering capacity and the extravesicular Ca2+ concentration in a manner that is consistent with a Ca2(+)-H+ exchange process. In the absence of a pH gradient, or the presence of a reversed gradient (basic interior), only background levels of cation uptake are detected. The driving force for A23187-mediated uptake of Ca2+ is shown to depend on the intravesicular proton pool rather than on a chelation process. This protocol provides a novel method for the efficient entrapment of high concentrations of Ca2+ and other cations in phospholipid vesicles.

P-31 nuclear magnetic resonance studies of the appearance of an isotropic component in dielaidoylphosphatidylethanolamine.

We have utilized phosphorus nuclear magnetic resonance, which provides an excellent means of characterizing the physical state of lipids, to investigate the polymorphic phase behavior of pure dielaidoylphosphatidylethanolamine (DEPE). We have observed a sharp isotropic component in the typical bilayer and inverted hexagonal P-31 NMR spectra. This component appears in the spectra of both the bilayer and inverted hexagonal lipid phases after several cycles through the bilayer-to-hexagonal phase transition. The magnitude of the isotropic component increased as a function of the number of cycles through the transition. The appearance of this component was not a function of time at constant temperature, but only a function of the number of cycles through the transition. The isotropic component is stable at all temperatures above the gel-to-liquid crystal transition, but it abruptly disappears when the lipid is cooled below the gel-to-liquid crystal phase transition. It is suggested that this isotropic phase is similar to the isotropic phase observed in dioleoylphosphatidylethanolamine (DOPE) by x-ray diffraction and identified as a cubic phase (Shyamsunder, E., S. M. Gruner, M. W. Tate, D. C. Turner, P. T. C. So, and C. P. S. Tilcock. 1988. Biochemistry. 27:2332-2336).

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine. Effect of n-alkanols.

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.

Effect of alcohols on the phase transitions of dihexadecylphosphatidylcholine.

We have systematically investigated the effect of short chain alcohols (methanol to n-propanol) on the phase transitions of 1,2-dihexadecylphosphatidylcholine (DHPC), a lipid that forms a stable interdigitated gel phase (L beta I) in aqueous solution. The temperature of the low-temperature L beta I to P beta' phase transition of DHPC was found to increase with alcohol concentration, showing that alcohol interacts preferentially with the interdigitated phase relative to the non-interdigitated gel. The main transition of DHPC exhibited a biphasic effect of alcohol concentration similar to that previously observed with DPPC (Rowe, E.S. (1983) Biochemistry 22,3299-3305). As alcohol concentration is increased the lower L beta I to P beta' and main P beta' to L alpha transitions of DHPC merge at the threshold concentration of the biphasic effect, so that above this concentration there is one phase transition from L beta I directly to L alpha. This is analogous to DPPC above its biphasic threshold. Similar to DPPC, the transition between L beta I and L alpha exhibits marked hysteresis.

Effect of n-alcohols and glycerol on the pretransition of dipalmitoylphosphatidylcholine.

We have systematically investigated the effect of short-chain n-alcohols and glycerol on the pretransition of 1,2-dipalmitoylphosphatidylcholine (DPPC) by spectrophotometry. It is found that the n-alcohols and glycerol remove the pretransition above a critical concentration for each ligand. In addition, the short-chain n-alcohols below the critical concentration decrease the pretransition temperature. The longer the aliphatic chain length of the n-alcohol (up to butanol) the greater the decrease in the pretransition temperature, and the lower the concentration necessary to remove the pretransition. However, glycerol differs from the short-chain n-alcohols in that it has no significant effect on either the pretransition or the main transition, but it is also capable of removing the pretransition above a critical concentration. It has previously been shown that alcohols have a biphasic effect on the main transition temperature of phosphatidylcholines (Rowe, E.S. (1983) Biochemistry 22, 3299-3305). At high alcohol concentrations, the main transition is not thermodynamically reversible (Rowe, E.S. (1985) Biochim. Biophys. Acta 813, 321-330). Recently, Simon and McIntosh (Biochim. Biophys. Acta (1984) 773, 169-172) have identified that at high ethanol concentration DPPC exists in the interdigitated phase. The critical ligand concentration at which the pretransition disappears coincides with the induction of main transition hysteresis and the biphasic alcohol effect in the main transition. These three effects appear to correlate with the induction of the interdigitated gel state by alcohols and glycerol.

The modulation of ion channels by the inhalation general anaesthetics. A1H-NMR investigation using unilamellar phospholipid membranes.

The modulation of a variety of mechanisms of channel-mediated transport across unilamellar phospholipid membranes by a range of halogenated inhalation general anaesthetics (chloroform, enflurane, halothane and methoxyflurane) was investigated using 1H-NMR spectroscopy. Transport of the probe ion Pr3+ across egg yolk phosphatidylcholine (PC) and dipalmitoyl phosphatidylcholine (DPPC) vesicular membranes in the presence of the channel forming polypeptides alamethicin 30 and melittin, and the polyene antibiotic nystatin, as well as the degree of vesicular lysis at the gel to liquid-crystal phase transition of DPPC vesicles was monitored. The observation that the inhalation general anaesthetics inhibit such membrane permeability independently of the channel system or type of lipid used, suggests that hydrogen-bonded water structure and/or hydrogen-bonding centres at dipolar lipid-polypeptide interfaces, can be likely sites of action of the general anaesthetics.

Phosphatidic acid regulates the activity of the channel-forming ionophores alamethicin, melittin, and nystatin: a 1H-NMR study using phospholipid membranes.

The regulation of ion channels by phosphatidic acid (a proposed active metabolite in the phosphatidylinositol effect) was investigated using 1H-NMR spectroscopy and small unilamellar phospholipid vesicles. Transport across egg-yolk phosphatidylcholine (egg PC) and dipalmitoyl phosphatidylcholine (DPPC) vesicular membranes in the presence of the channel-forming ionophores alamethicin, melittin, and nystatin was monitored using the lanthanide probe ion Pr3+. In the absence of the ionophores, phosphatidic acid (PA) alone was found to have no ionophore properties, but in the presence of the ionophores the incorporation of 3 mol % phosphatidic acid in the bilayer markedly increased the rate of transport using melittin and nystatin, but decreased the rate using alamethicin, independent of the type of phosphatidylcholine used. The presence of PA in the bilayer also stimulated the production of lytic type channels, the extent of which were both ionophore- and lipid-dependent. These results are discussed in terms of possible molecular interactions between the PA, the individual ionophores, and type of lipid used.