Maureen M. Momsen

Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol.

The effect of cholesterol on the interfacial elastic packing interactions of various molecular species of phosphatidylcholines (PCs) has been investigated by using a Langmuir-type film balance and analyzing the elastic area compressibility moduli (Cs(-1)) as a function of average cross-sectional molecular area. Emphasis was on the high surface pressure regions (pi > or = 30 mN/m) which are thought to mimic biomembrane conditions. Increasing levels of cholesterol generally caused the in-plane elasticity of the mixed monolayers to decrease. Yet, the magnitude of the cholesterol-induced changes was markedly dependent upon PC hydrocarbon structure. Among PC species with a saturated sn-1 chain but different sn-2 chain cis unsaturation levels [e.g., myristate (14:0), oleate (18:1delta9(c), linoleate (18:2delta9,12(c), arachidonate (20:4delta5,8,11,14(c), or docosahexenoate (22:6delta4,7,10,13,16,19(c)], the in-plane elasticity moduli of PC species with higher sn-2 unsaturation levels were less affected by high cholesterol mol fractions (e.g., >30 mol %) than were the more saturated PC species. The largest cholesterol-induced decreases in the in-plane elasticity were observed when both chains of PC were saturated (e.g., di-14:0 PC). When both acyl chains were identically unsaturated, the resulting PCs were 20-25% more elastic in the presence of cholesterol than when their sn-1 chains were long and saturated (e.g., palmitate). The mixing of cholesterol with PC was found to diminish the in-plane elasticity of the films beyond what was predicted from the additive behavior of the individual lipid components apportioned by mole and area fraction. Deviations from additivity were greatest for di-14:0 PC and were least for diarachidonoyl PC and didocosahexenoyl PC. In contrast to Cs(-1) analyses, sterol-induced area condensations were relatively unresponsive to subtle structural differences in the PCs at high surface pressures. Cs(-1) versus average area plots also indicated the presence of cholesterol concentration-dependent, low-pressure (<14 mN/m) phase boundaries that became more prominent as PC acyl chain unsaturation increased. Hence, area condensations measured at low surface pressures often do not accurately portray which lipid structural features are important in the lipid-sterol interactions that occur at high membrane-like surface pressures.

Sphingomyelin Interfacial Behavior: The Impact of Changing Acyl Chain Composition ☆

Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) at the end of C3-, C5-, C7-, or C9-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me4-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me4-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me4-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and ∼506–515 nm) but also showed the absence of the 620–630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me4-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

Physical and photophysical characterization of a BODIPY phosphatidylcholine as a membrane probe.

Lipids containing the dimethyl BODIPY fluorophore are used in cell biology because their fluorescence properties change with fluorophore concentration (C.-S. Chen, O. C. Martin, and R. E. Pagano. 1997. Biophys J. 72:37-50). The miscibility and steady-state fluorescence behavior of one such lipid, 1-palmitoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (PBPC), have been characterized in mixtures with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). PBPC packs similarly to phosphatidylcholines having a cis-unsaturated acyl chain and mixes nearly ideally with SOPC, apparently without fluorophore-fluorophore aggregation. Increasing PBPC mole fraction from 0.0 to 1.0 in SOPC membranes changes the emission characteristics of the probe in a continuous manner. Analysis of these changes shows that emission from the excited dimethyl BODIPY monomer self quenches with a critical radius of 25.9 A. Fluorophores sufficiently close (< or =13.7 A) at the time of excitation can form an excited dimer, emission from which depends strongly on total lipid packing density. Overall, the data show that PBPC is a reasonable physical substitute for other phosphatidylcholines in fluid membranes. Knowledge of PBPC fluorescence in lipid monolayers has been exploited to determine the two-dimensional concentration of SOPC in unilamellar, bilayer membranes.

Sterol structure and sphingomyelin acyl chain length modulate lateral packing elasticity and detergent solubility in model membranes.

Membrane microdomains, such as caveolae and rafts, are enriched in cholesterol and sphingomyelin, display liquid-ordered phase properties, and putatively function as protein organizing platforms. The goal of this investigation was to identify sterol and sphingomyelin structural features that modulate surface compression and solubilization by detergent because liquid-ordered phase displays low lateral elasticity and resists solubilization by Triton X-100. Compared to cholesterol, sterol structural changes involved either altering the polar headgroup (e.g., 6-ketocholestanol) or eliminating the isooctyl hydrocarbon tail (e.g., 5-androsten-3beta-ol). Synthetic changes to sphingomyelin resulted in homogeneous acyl chains of differing length but of biological relevance. Using a Langmuir surface balance, surface compressional moduli were assessed at various surface pressures including those (pi > or =30 mN/m) that mimic biomembrane conditions. Sphingomyelin-sterol mixtures generally were less elastic in a lateral sense than chain-matched phosphatidylcholine-sterol mixtures at equivalent high sterol mole fractions. Increasing content of 6-ketocholestanol or 5-androsten-3beta-ol in sphingomyelin decreased lateral elasticity but much less effectively than cholesterol. Our results indicate that cholesterol is ideally structured for maximally reducing the lateral elasticity of membrane sphingolipids, for enabling resistance to Triton X-100 solubilization, and for interacting with sphingomyelins that contain saturated acyl chains similar in length to their sphingoid bases.

Interactions of olopatadine and selected antihistamines with model and natural membranes.

Objective: Olopatadine, an effective topical ocular human conjunctival mast cell stabilizer/antihistaminic antiallergic drug, was evaluated and compared to selected classical antihistamines for their interaction with model and natural membranes to ascertain potential functional consequences of such interactions. Methods: The model membranes examined consisted of the argon-buffer interface and monomolecular films of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) at the argon-buffer interface. Interactions with the model membranes were detected as changes in surface tension, i.e., surface pressure. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage, hemoglobin release, lactate dehydrogenase release, and histamine release from appropriate cell types. Results: Measurements at the argon-buffer interface revealed intrinsic surface activity for all agents that ranged from highly surface-active to weakly surface-active in the order of: desloratadine > clemastine > azelastine = ketotifen > diphenhydramine > pyrilamine > emedastine > epinastine =olopatadine. This order of amphipathic behavior was confirmed for most of the compounds by estimates of their dissociation constants (K d,L ) determined from interactions with SOPC monolayers adjusted to a surface pressure approximating that of natural membranes. Epinastine was the only antihistamine that showed a disproportionately greater increase in surface activity toward SOPC in monolayer when compared to other antihistamines. Dissociation constants could not be established for olopatadine because of its low affinity for both the argon-buffer interface and the SOPC monolayer. Functional consequences of these interactions were assessed with natural membranes by 6-carboxyfluorescein leakage (erythrocyte ghosts), hemoglobin release (erythrocytes), lactate dehydrogenase release (conjunctival mast cells, corneal epithelial cells), and histamine release (conjunctival mast cells). Aside from olopatadine and emedastine, all antihistamines promoted a concentration- dependent leakage of hemoglobin from intact erythrocytes. The concentration of drug required to cause half-maximal hemoglobin release (H 50 ) from erythrocytes correlated linearly (r = 0.98) with the SOPC dissociation constants (K d,L ) estimated for the different antihistaminic agents interacting with SOPC monolayers.A similarly high correlation (r = 0.85) emerged from a plot with a slope approaching unity that related drug concentrations required for half-maximal hemoglobin leakage from erythrocytes to threshold doses of drug that caused histamine release from human conjunctival mast cells. Olopatadine was the only agent that did not promote membrane perturbation as monitored by either hemoglobin release from intact erythrocytes, LDH release from human conjunctival mast cells, or 6-carboxyfluorescein release from erythrocyte ghosts. Assessment of the lytic potential of marketed concentrations of ketotifen (0.025%), azelastine (0.05%), and epinastine (0.05%) revealed significant membrane perturbation of human conjunctival mast cells and, importantly, human corneal epithelial cells as indexed by LDH release. This was in contrast to marketed concentrations of olopatadine (0.1%) which maintained normal mast cell and corneal epithelial cell membrane function. Conclusions: Combined, these results support the notion that the disruption of natural cell membranes by surface-active antihistamines occurs not through a receptor-mediated process, but is the consequence of a direct interaction of these agents with the cell membrane. This is corroborated by surface pressure-concentration isotherms for adsorption of five different antihistaminic agents to SOPC monolayers where 50% lysis occurred at a surface pressure of 42.9 ± 1.1mN/m. Olopatadine appears to be unique among the agents tested by demonstrating low intrinsic surface activity, thus limiting its interaction with natural membranes. At concentrations of about half-maximal compound solubility (i.e., 5.0mM or a 0.19% drug solution), olopatadine generated SOPC monolayer surface pressures (i.e., 39.82 ± 0.10 mN/m) that were below those that promoted membrane perturbation and onset of hemoglobin leakage. Olopatadine’s restricted interaction with membrane phospholipids limits the degree of membrane perturbation and release of intracellular constituents, including histamine, LDH,and hemoglobin, which is believed to contribute to olopatadine’s topical ocular comfort and patient acceptance.

Acyl Chain-Length Asymmetry Alters the Interfacial Elastic Interactions of Phosphatidylcholines

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.

Packing and Electrostatic Behavior of sn-2-Docosahexaenoyl and -Arachidonoyl Phosphoglycerides

Mammalian synaptic membranes appear to contain high proportions of specific, sn-1-stearoyl-2-docosahexaenoyl- and sn-1-stearoyl-2-arachidonoyl phosphoglycerides, but the structural significance of this is unclear. Here we used a standardized approach to compare the properties of homogeneous monolayers of the corresponding phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidic acids with those of control monolayers of sn-1-stearoyl-2-oleoyl- and sn-1-palmitoyl-2-oleoyl phosphoglycerides. Major findings were: 1), that the presence of an sn-2-docosahexaenoyl group or an sn-2-arachidonoyl group increases the molecular areas of phosphoglycerides by 3.8 A(2) (7%) relative to the presence of an sn-2-oleoyl group; 2), that the phosphorylcholine headgroup independently increases molecular areas by a larger amount, 7.1 A(2) (13%); and 3), that the dipole moments of species having an arachidonoyl moiety or an oleoyl moiety are 83 mD (19%) higher than those of comparable docosahexaenoic acid-containing phosphoglycerides. These and other results provide new information about the molecular packing properties of polyenoic phosphoglycerides and raise important questions about the role of these phosphoglycerides in synapses.

Lipid Lateral Organization in Fluid Interfaces Controls the Rate of Colipase Association

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.

The affinities of procolipase and colipase for interfaces are regulated by lipids

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.