Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.     

Effect of cholesterol on Ca2+-induced aggregation of liposomes and calcium diphosphatidate membrane traversal

Sonicated cholesterol-phosphatidylcholine (PC) liposomes containing 4 mol % phosphatidic acid (PA) aggregate in 10 mM Ca2+, slowly at low molar fractions of cholesterol (up to 30%) and 15 times faster at higher concentrations; the inflection point is at ca. 35 mol % bilayer cholesterol. O-[[(Methoxyethoxy)ethoxy]ethyl]cholesterol (OH-blocked cholesterol) does not give this rate enhancement. If PC is replaced by diether PC (CO groups abolished), cholesterol does not accelerate aggregation at concentrations in the bilayer below 50 mol %. No change in Ca2+-induced aggregation rates was observed if the ester CO groups of the bridge-forming PA only were replaced by CH2 (diether PA) in liposomes containing PC and cholesterol. PA-mediated Ca2+ membrane traversal seems to be accelerated by the addition of cholesterol to the PC-PA membrane, but analysis shows that the effect is due to the bilayer condensation effect of cholesterol resulting in an increase in the surface concentration of PA and that membrane cholesterol in fact slightly reduces the rate of Ca(PA)2 traversal; OH-blocked cholesterol, however, increases this rate 3-fold. It appears that lipid OH and CO groups interact, directly or with the mediation of water, in establishing the structure of the membrane "hydrogen belts", i.e., the strata containing those hydrogen-bond donors and acceptors. Cholesterol hydroxyl above 33 mol % (saturation of a 2:1 PC/cholesterol complex?) causes a restructuring of the hydrogen belts that facilitates membrane-water-membrane dehydration, the prerequisite for liposome aggregation by trans-Ca(PA)2 formation. On the other hand, the formation of the dehydrated cis-Ca(PA)2 complex that precedes Ca2+ membrane traversal is not accelerated by presence of the cholesterol hydroxyl group.     

Ca(phosphatidate)2 can traverse liposomal bilayers

Phosphatidic acid can act as Ca2+ cross-membrane ionophore without the necessity of previous autoxidation. The apparent PA-CA2+ dissociation constant is 3 X 10(-3), i.e., in the range of extracellular Ca2+ concentration. There is at least 100-fold preference for Ca2+ over Mg2+. Ca2+ transfer rates are proportional to the square of phosphatidic acid concentration in the bilayer. Removal of the fatty acid ester CO groups reduces the Ca2+ ferrying rate by more than 90 percent. It appears that the cation is held in a cage formed by phosphate and carbonyl oxygens of two PA molecules. In this coordination complex both Ca2+ and the phosphatidic acid headgroups are dehydrated, and the Ca(phosphatide)2 assembly becomes lipid-soluble and can traverse the bilayer.     

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.     

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid containing bilayers

The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations.     

Behavioral differences between phosphatidic acid and phosphatidylcholine in the presence of the nicotinic acetylcholine receptor

It has been found experimentally that negatively charged phosphatidic acid (PA) lipids and cholesterol molecules stabilize the nicotinic acetylcholine receptor (nAChR) in a functional resting state that can participate in an agonist-induced conformational change. In this study, we compare phosphatidylcholine (PC) and PA lipid behavior in the presence of the nAChR to determine why PC lipids do not support a functional nAChR. For lipids that are located within 1.0 nm of the protein, both PC and PA lipids have very similar order-parameter and bilayer-thickness values, which indicate that the annular lipid properties are protein-dependent. The most significant difference between the PC and PA bilayers is the formation of a lipid domain around the protein, which is visible in the PA bilayer but not the PC bilayer. This suggests that the PA domain may help stabilize the nAChR resting state. The PA lipids in the microdomain have a decreased order compared to a homogeneous PA bilayer and the lipids near the protein attempt to increase the free space in their vicinity by residing in multiple lateral planes.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mechanism for phosphoglyceride and Ca2+ transbilayer movement

The ionophoretic capabilities of phosphoglycerides (PL) have been examined by measuring their translocation via cations from aqueous dispersions into linear and cyclic hydrocarbons. The PL surveyed were phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI). Only PA displayed ionophoretic activity in single lipid dispersions with a cation selectivity order of Mn greater than Ca. PG, PE and PC, but not PI, had a synergistic affect of PA induced translocation. These PL, inactive individually or in any combination, became strong Ca2+ ionophores of variable activity in association with PA. A dimeric structure proposed for the ionophoretic species forms the basis of a mechanism for transbilayer movement of PA, PG, PE and PC which would establish an asymmetric distribution of these lipids in the two faces of the bilayer by equilibrium processes.     

Phospholipase D in homogenates from HL-60 granulocytes: implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.     

Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor

When isolated frog (Rana catesbeiana) rod outer segment (ROS) fragments were incubated with [gamma-32P]ATP in the dark, only two of phospholipids, i.e., phosphatidylinositol-4-phosphate (DPI) and phosphatidic acid (PA) incorporated 32P. Upon addition of DPI (100 microM), considerable amount of 32P was incorporated into phosphatidylinositol-4,5-bisphosphate (TPI) as well as DPI and PA. Exposure of the ROS membranes to 5 sec flash of light resulted in approx. 20% decrease in the labeled TPI, while no significant effect was observed on DPI and PA. It was also observed that Ca2+ markedly accelerated the production of PA in the dark, while it reduced the 32P-incorporation into TPI. These results suggest that there is light- and/or Ca2+-dependent TPI-specific phospholipase C in ROS of vertebrate photoreceptors.     

Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.     

Oxidative stability of polyunsaturated fatty acid in phosphatidylcholine liposomes

Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC + 1,2-diLA-PC, or 1,2-diPA-PC + 1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC + diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC + diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.     

Can the stereoselective effects of the anesthetic isoflurane be accounted for by lipid solubility?

Isoflurane is an inhalational general anesthetic widely used in surgical operations as a racemic mixture of its two optical isomers. The recent availability of pure enantiomers of isoflurane has encouraged their use in experimental studies, and stereoselective effects have now been observed on anesthetic-sensitive neuronal ion channels. Although it has been assumed that such chiral effects demonstrate direct interactions with proteins, it is possible that they could be due to stereoselective interactions with chiral membrane lipids. We have determined the partition coefficients of the two optical isomers of isoflurane between lipid bilayers and water, using racemic isoflurane and gas chromatography with a chiral column. For lipid bilayers of phosphatidylcholine (PC) and 4 mol% phosphatidic acid (PA), both with and without cholesterol (CHOL), we found equal partitioning of the isoflurane optical isomers. The ratios of the S(+) to R(-) isoflurane partition coefficients were (mean +/- SEM): 1.018 +/- 0.010 for bilayers of PC/CHOL/PA (mole ratios 56:40:4) and 1.011 +/- 0.002 for bilayers of PC/PA (mole ratio 96:4). Molar partition coefficients for racemic isoflurane were 49 +/- 4 and 165 +/- 10, respectively. These findings support the view that the stereoselective effects on ion channels observed with isoflurane are due to direct actions on proteins rather than lipids.     

Oxidative Stability of Polyunsaturated Fatty Acid in Phosphatidylcholine Liposomes

Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC+1,2-diLA-PC, or 1,2-diPA-PC+1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC+diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC+diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.     

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.     

Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor

Bilayers containing phosphatidylcholine (PC) and the anionic lipid phosphatidic acid (PA) are particularly effective at stabilizing the nicotinic acetylcholine receptor (nAChR) in a functional conformation that undergoes agonist-induced conformational change. The physical properties of PC membranes containing PA are also substantially altered upon incorporation of the nAChR. To test whether or not the negative charge of PA is responsible for this "bi-directional coupling," the nAChR was reconstituted into membranes composed of PC with varying levels of the net negatively charged lipid phosphatidylserine (PS). In contrast to PA, increasing levels of PS in PC membranes do not stabilize an increasing proportion of nAChRs in a functional resting conformation, nor do they slow nAChR peptide hydrogen exchange kinetics. Incorporation of the nAChR had little effect on the physical properties of the PC/PS membranes, as monitored by the gel-to-liquid crystal phase transition temperatures of the bilayers. These results show that a net negative charge alone is not sufficient to account for the unique interactions that occur between the nAChR and PC/PA membranes. Incorporation of the receptor into PC/PS membranes, however, did lead to an altered head group conformation of PS possibly by recruiting divalent cations to the membrane surface. The results show that the nAChR has complex and unique interactions with both PA and PS. The interactions between the nAChR and PS may be bridged by divalent cations, such as calcium.     

Enhanced apoptosis through farnesol inhibition of phospholipase D signal transduction

Farnesol is a catabolite of the cholesterol biosynthetic pathway that preferentially causes apoptosis in tumorigenic cells. Phosphatidylcholine (PC), phosphatidic acid (PA), and diacylglycerol (DAG) were able to prevent induction of apoptosis by farnesol. Primary alcohol inhibition of PC catabolism by phospholipase D augmented farnesol-induced apoptosis. Exogenous PC was unable to prevent the increase in farnesol-induced apoptosis by primary alcohols, whereas DAG was protective. Farnesol-mediated apoptosis was prevented by transformation with a plasmid coding for the PA phosphatase LPP3, but not by an inactive LPP3 point mutant. Farnesol did not directly inhibit LPP3 PA phosphatase enzyme activity in an in vitro mixed micelle assay. We propose that farnesol inhibits the action of a DAG pool generated by phospholipase D signal transduction that normally activates an antiapoptotic/pro-proliferative target.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.