Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients

Transmembrane movement of dopamine in response to K+ or H+ ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K+ diffusion potential (delta psi; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30-40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting delta pH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K+ potential is more complex. Although generation of a K+ diffusion potential results in acidification of the vesicle interior, the magnitude of the induced delta pH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (greater than 200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium.     

Small and large unilamellar vesicle membranes as model system for bile acid diffusion in hepatocytes.

Uptake of bile acids into the liver cell occurs via active transport or passive diffusion. In a model system, passive diffusion was studied in liposomes using pyranine fluorescence. Rate constants for the diffusion of diverse more polar or more apolar bile acids were examined. Hydrophobic lithocholic acid (LCA) revealed a maximal rate constant of 0.057 s(-1); with the polar ursodeoxycholic acid (UDCA), the value was 0.019 s(-1). UDCA (3 mol%) effectively decreased the rate constant of 0.1 mM chenodeoxycholic acid (CDCA), whereas cholesterol reached a similar decrease only between 5 and 10 mol%. At higher concentrations of CDCA (above 1 mM) or LCA (0.3-0.4 mM), breaking up of liposomal structure was confirmed by light-scattering decrease and increase of carboxyfluorescein fluorescence. Changes in lipid composition of phosphatidylcholine (PC)- small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) also caused decreasing rate constants. For a cardiolipin (CL):PC ratio of 1:20 the CDCA (0.1 mM) rate constant was 71% lower (0.015 s(-1)) and for a sphingomyelin (SM):PC ratio of 2:1 the rate constant was 50% lower (0.026 s(-1)). Changes in membrane fluidity were detected using membrane anisotropy measurements with the 1,6-diphenyl-1,3, 5-hexatriene (DPH) method. Membrane fluidity was reduced with cholesterol- but not with CL- or SM-containing SUVs (ratio: cholesterol, CL, SM:PC of 1:5). This model system is currently used for the analysis of more complex lipid vesicles resembling the plasma/hepatocyte membrane, which is either stabilized or destabilized by appropriate conditions. The results should become clinically relevant.     

Modeling of matrix vesicle biomineralization using large unilamellar vesicles

Stable, large unilamellar vesicles (LUV) have been constructed that model matrix vesicles (MV) in inducing de novo mineral formation when incubated in synthetic cartilage lymph (SCL). Using a dialysis method for incorporation of predetermined pure lipid, electrolyte and protein constituents, the detergent n-octyl beta-D-glucopyranoside enabled formation of stable, impermeable LUV with a diameter ( approximately 300 nm), lipid composition (phosphatidylcholine-phosphatidylserine-cholesterol, 7:2:2, molar ratio) and enclosed inorganic phosphate level (25-100 mM) similar to that of native MV. Mineral formation by these LUVs was measured by 45Ca(2+) uptake and FTIR analysis following incubation in SCL. Addition of the ionophore A23187 to SCL enabled 45Ca(2+) uptake comparable to that of native MV. FTIR analysis revealed that crystalline mineral formed in the LUV during incubation in SCL, but not in the absence of ionophore. This mineral had an IR absorption spectrum like that of the acid-phosphate-rich, octacalcium phosphate-like mineral formed by native MV. Perturbing the LUV membrane with either detergents or phospholipase A(2) following prior incubation in SCL enabled egress of mineral crystallites from the vesicle lumen, stimulating further mineral formation. Annexin V, a major protein in native MV with known Ca(2+) channel activity, incorporated into the LUV lumen or added to the external medium, induced only limited 45Ca(2+) uptake. This indicates that additional factors are required for annexin V to form Ca(2+) channels. Nevertheless for the first time, stable LUVs have been constructed with MV-like lipid, electrolyte, and protein composition and size that induce formation of mineral like that formed by native MV.     

Nuclear magnetic resonance study of acetic acid permeation of large unilamellar vesicle membranes.

The permeation of acetic acid through large unilamellar phospholipid vesicle membranes has been investigated using the unique capability of nuclear magnetic resonance to characterize flow under pseudo-equilibrium conditions. Two types of experiments have been employed: total line shape analysis and selective population transfer. These techniques are sensitive to permeation on time scales ranging form 0.001 to 10.0 s. The permeation rate dependence on pH and acetic acid concentration indicates that the neutral acetic acid monomer is the dominant permeant species with a permeation coefficient of 5 +/- 2 x 10-4 cm/s. Mechanisms of permeation and the applicability of nuclear magnetic resonance methodology are discussed.     

Phosphatidic acid releases calcium from a platelet membrane fraction in vitro

A platelet membrane fraction which actively sequesters calcium in the presence of ATP was prepared and the influence of phosphatidic acid evaluated. At 10–60 μg/ml phosphatidic acid caused a concentration dependent release of calcium from the membrane fraction. The calcium was released from inside the vesicles, since release occurred in the presence of EGTA used to bind calcium outside the membrane vesicles. Aspirin failed to inhibit release of calcium by phosphatidic acid. Our results may explain, in part, the prostaglandin and thromboxane independent calcium release which occurs in response to certain aggregating agents. Thus, phosphatidic acid, or a metabolite, may have an important role intracellularly in platelets in promoting calcium movement.     

Phosphatidic acid induces calcium influx in neutrophils via verapamil-sensitive calcium channels

Phosphatidic acid (PA) induces a biphasic Ca(2+) mobilization response in human neutrophils. The initial increase is due to the mobilization of Ca(2+) from intracellular stores, whereas the secondary increase is due to the influx of Ca(2+) from extracellular sources. The present investigation characterizes PA-induced Ca(2+) influx in neutrophils. Depolarization of neutrophils by 50 mM KCl enhanced PA-induced Ca(2+) influx, whereas verapamil, a Ca(2+) channel blocker, attenuated this response in a dose-dependent manner. These observations suggest that PA-induced Ca(2+) influx is mediated via verapamil-sensitive Ca(2+) channels. Stimulation of neutrophils with exogenous PA results in accumulation of endogenously generated PA with a time course similar to the effects of exogenous PA on Ca(2+) influx. Ethanol inhibited the accumulation of endogenous PA and calcium mobilization, indicating that activation of membrane phospholipase D plays a role in PA-mediated Ca(2+) influx. The results of this study suggest that exogenously added PA stimulates the generation of intracellular PA, which then mediates Ca(2+) influx through verapamil-sensitive Ca(2+) channels.     

Characterization of ionomycin as a calcium ionophore.

The ionophorous properties of a new antibiotic, ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.     

Protection of large unilamellar vesicles by trehalose during dehydration: retention of vesicle contents

The ability of trehalose and other sugars to maintain the integrity of large unilamellar vesicles subjected to dehydration and rehydration has been investigated. It is shown, employing freeze-fracture techniques, that large unilamellar vesicles prepared in the presence of trehalose at 125 mM or higher concentration do not exhibit significant structural changes during the dehydration-rehydration cycle. Further, up to 90% of entrapped 22Na or [3H]inulin is retained during this process. Other sugars also exhibited similar protective effects where trehalose was most effective, followed by sucrose, maltose, glucose and lactose. It is demonstrated that proton or Na+/K+ electrochemical gradients can be maintained during the dehydration-rehydration process, which can subsequently be used to drive the uptake of lipophilic cationic drugs such as adriamycin. The implications for long-term storage of liposomal systems for use in drug-delivery protocols are discussed.     

Phosphatidic acid-induced calcium mobilization in osteoblasts

Phosphatidic acid (PA) evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i) in osteoblasts isolated from neonatal mouse calvaria. This increase was observed in both low (below 150 microM) and high (1.26 mM) Ca2+-containing medium. In contrast, other phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, failed to increase [Ca2+]i in osteoblasts. In high Ca2+-containing medium, A23187 also increased [Ca2+]i in the cells, but the mode of the change was different from that in the case of PA. These results suggest that PA may induce Ca2+-mediated cellular responses through Ca2+ release from intracellular stores in osteoblasts.     

Large vesicle contamination in small,unilamellar vesicles

Small, unilamellar phospholipid vesicles have been prepared using a new, high-powdered cup sonifier that avoids contact of the sample with a titanium probe. These vesicles have been characterized by gel filtration chromatography both before and after fractionation by high-speed centrifugation. Plots of the turbidity of centrifuged vesicles between 300 and 650 nm against the reciprocal fourth power of the scattering wavelength were linear with zero intercepts (extrapolated to infinite wavelength). In the presence of minute quantities of large, multilamellar vesicles, these plots remained linear but had intercepts quantitatively proportional to the amount of contaminating large vesicles. Since this measurement requires only a standard spectrophotometer and very small quantities of lipid, this method is suggested as a useful assay for determining contamination of small vesicle preparations by large vesicles. Two applications of this method as well as a practical limitation are discussed.     

Phosphatidic acid synthesis in bacteria

Membrane phospholipid synthesis is a vital facet of bacterial physiology. Although the spectrum of phospholipid headgroup structures produced by bacteria is large, the key precursor to all of these molecules is phosphatidic acid (PtdOH). Glycerol-3-phosphate derived from the glycolysis via glycerol-phosphate synthase is the universal source for the glycerol backbone of PtdOH. There are two distinct families of enzymes responsible for the acylation of the 1-position of glycerol-3-phosphate. The PlsB acyltransferase was discovered in Escherichia coli, and homologs are present in many eukaryotes. This protein family primarily uses acyl–acyl carrier protein (ACP) endproducts of fatty acid synthesis as acyl donors, but may also use acyl-CoA derived from exogenous fatty acids. The second protein family, PlsY, is more widely distributed in bacteria and utilizes the unique acyl donor, acyl-phosphate, which is produced from acyl-ACP by the enzyme PlsX. The acylation of the 2-position is carried out by members of the PlsC protein family. All PlsCs use acyl-ACP as the acyl donor, although the PlsCs of the γ-proteobacteria also may use acyl-CoA. Phospholipid headgroups are precursors in the biosynthesis of other membrane-associated molecules and the diacylglycerol product of these reactions is converted to PtdOH by one of two distinct families of lipid kinases. The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest that these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Phosphatidic acid synthesis in yeast

1. The integrity of DNA extracted from Escherichia coli strain 15T(-) after thymine-less death was examined by studying the effects of treatment with aqueous alkali on the solubility in dilute acids and by viscosity and ultracentrifugal measurements, some of which were designed to detect single-strand breaks or inter-strand cross-links. None of the results showed that there was any modification or damage associated with thymine-less death.     

Phosphatidic acid activates a wound-activated MAPK in Glycine max

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.     

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.     

Phosphatidic acid metabolism,calcium ions and transmitter release from electrically stimulated synaptosomes

Synaptosomes isolated from guinea pig brain cortex were stimulated electrically in a medium containing [32P]-orthophosphate. The electrical stimulation caused increased labelling of phosphatidic acid in a synaptic vesicle fraction prepared by osmotic shock of the incubated synaptosomes. Electrical stimulation also provokes transmitter release from the synaptosomes. Both increased phosphatidate labelling and transmitter release required calcium ions in the medium. The effects are discussed in relation to earlier work with acetylcholine and the possible involvement of membrane phosphatidic acid in transmitter release by exocytosis.     

Phosphatidic acid synthesis in Escherichia coli

The kinetic properties of acyl-coenzyme A (CoA): l-alpha-glycerol-phosphate trans-acylase (EC 2.3.1.15) from Escherichia coli were studied. At 10 C, a temperature at which the reaction was proportional to time and enzyme concentration, the enzyme had an apparent K(m) of 60 mum for l-alpha-glycerol-phosphate. The curve describing the velocity of the reaction as a function of palmitoyl-CoA concentration was sigmoid but the plot of v(-1) versus [S](-3) gave a straight line. A K(m) of about 11 mum was calculated for palmitoyl-CoA. Adenosine triphosphate specifically inhibited the reaction, being a noncompetitive inhibitor in respect to l-alpha-glycerol phosphate. Inhibition only occurred with high concentrations of palmitoyl-CoA, and maximal inhibition was 60%.     

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis

Specific cellular components have been identified to function in abscisic acid (ABA) regulation of stomatal apertures, including calcium, the cytoskeleton, and phosphatidic acid. In this study, the regulation and dynamic organization of microtubules during ABA-induced stomatal closure by phospholipase D (PLD) and its product PA were investigated. ABA induced microtubule depolymerization and stomatal closure in wide-type (WT) Arabidopsis, whereas these processes were impaired in PLD mutant (pldα1). The microtubule-disrupting drugs oryzalin or propyzamide induced microtubule depolymerization, but did not affect the stomatal aperture, whereas their co-treatment with ABA resulted in stomatal closure in both WT and pldα1. In contrast, the microtubule-stabilizing drug paclitaxel arrested ABA-induced microtubule depolymerization and inhibited ABA-induced stomatal closure in both WT and pldα1. In pldα1, ABA-induced cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) elevation was partially blocked, and exogenous Ca²⁺-induced microtubule depolymerization and stomatal closure were impaired. These results suggested that PLDα1 and PA regulate microtubular organization and Ca²⁺ increases during ABA-induced stomatal closing and that crosstalk among signaling lipid, Ca²⁺, and microtubules are essential for ABA signaling.     

Phosphatidic acid: a multifunctional stress signaling lipid in plants

Phosphatidic acid (PA) has only recently been identified as an important signaling molecule in both plants and animals. Nonetheless, it already promises to rival the importance of the classic second messengers Ca(2+) and cAMP. In plants, its formation is triggered in response to various biotic and abiotic stress factors, including pathogen infection, drought, salinity, wounding and cold. In general, PA signal production is fast (minutes) and transient. Recently, our understanding of the role of PA formation in stress responses as a result of phospholipases C and D activity has greatly increased. Moreover, the first protein targets of PA have been identified. Based on this recent work, potential mechanisms by which PA provokes downstream effects are emerging.     

The role of calcium in eicosanoid production induced by ricinoleic acid or the calcium ionophore A23187

Rat isolated intestine incubated in Krebs solution converted exogenous [14C]-arachidonic acid into products that chromatographed with prostaglandins, leukotriene B4 and 5-hydroxy-eicosatetraenoic acid. Accumulation of these products was increased by the laxative ricinoleic acid (0.34 mM) or the calcium ionophore A23187 (7.6 microM). In the presence of the calcium antagonists TMB-8 (0.43 microM) or verapamil (0.2 microM) the mean effects of ricinoleic acid or the calcium ionophore were smaller. Stimulation of arachidonic acid metabolism by ricinoleic acid therefore seems likely to involve a calcium-dependent mechanism.