Burton J. Litman

Mechanisms of action of docosahexaenoic acid in the nervous system

AbtractThis review describes (from both the animal and human literature) the biological consequences of losses in nervous system docosahexaenoate (DHA). It then concentrates on biological mechanisms that may serve to explain changes in brain and retinal function. Brief consideration is given to actions of DHA as a nonesterified fatty acid and as a docosanoid or other bioactive molecule. The role of DHA-phospholipids in regulating G-protein signaling is presented in the context of studies with rhodopsin. It is clear that the visual pigment responds to the degree of unsaturation of the membrane lipids. At the cell biological level, DHA is shown to have a protective role in a cell culture model of apoptosis in relation to its effects in increasing cellular phosphatidylserine (PS); also, the loss of DHA leads to a loss in PS. Thus, through its effects on PS, DHA may play an important role in the regulation of cell signaling and in cell proliferation. Finally, progress has been made recently in nuclear magnetic responance studies to delineate differences in molecular structure and order in biomembranes due to subtle changes in the degree of phospholipid unsaturation.

A ROLE FOR PHOSPHOLIPID POLYUNSATURATION IN MODULATING MEMBRANE PROTEIN FUNCTION

Visual transduction is one of the best characterized G protein—coupled signalling systems. In addition, about 50% of the disk membrane phospholipid acyl chains are 22:6n-3, making this system ideal for determining the role of polyunsaturation in modulating membrane-signalling systems. The extent of formation of metarhodopsin II (MII), the G protein—activating photointermediate of rhodopsin, was studied in phospholipid vesicles composed of a variety of phosphatidylcholines, differing in their acyl chain composition at thesn-2 position. The amount of MII formed increased progressively with the level of acyl chain unsaturation at thesn-2 position. The effect of added cholesterol was to reduce the amount of MII formed. The acyl chain packing free volume of the rhodopsin containing lipid vesicles was characterized by a fractional volume parameter fv derived from measurements of the time-resolved fluorescence anisotropy decay of the hydrophobic membrane probe 1,6-diphenyl-1,3,5-hexatriene. The relationship amongsn-2 acyl chain unsaturation, cholesterol content, and MII formation is explained on the basis of variation in fv with bilayer lipid composition and a novel model for the packing of phospholipids containing polyenoic acyl chains, such as 22:6n-3.

The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways

In order to understand the role of the high levels of docosahexaenoic acid (DHA) in neuronal and retinal tissue, a study of the effect of membrane lipid composition on the visual pathway, a G protein-coupled system, was undertaken. The level of metarhodopsin II (MII) formation was determined to be a function of phospholipid acyl-chain unsaturation, with the highest levels seen in DHA-containing bilayers. Similarly, the rate of coupling of MII to the retinal G protein, Gt, to form a MII-Gt complex, was enhanced in DHA bilayers relative to less unsaturated phospholipids. Complex formation initiates the first stage of amplification in the visual pathway. The activation of the cGMP phosphodiesterase (PDE), the effector enzyme, represents the integrated pathway function. DHA-containing bilayers were found to support PDE levels comparable to those of the rod outer segment (ROS) disk membranes. Inclusion of 30 mol cholesterol in the reconstituted bilayers had an inhibitory effect on each step in the visual pathway studied. Inclusion of cholesterol reduced MII formation and PDE activity and increased the lag time between the appearance of MII and the formation of the MII-Gt complex. However, signaling in DHA bilayers was far less affected by the addition of cholesterol than in bilayers containing less unsaturated phospholipids. These studies point up the importance of DHA acyl chains in promoting optimal function in G protein-coupled signaling pathways. The results reported here suggest that visual and cognitive deficits observed in n-3 deficiency may result from decreased efficiency in related neurotransmitter and visual signaling pathways in the absence of DHA.

Determination of membrane cholesterol partition coefficient using a lipid vesicle-cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition.

Lateral domain or raft formation in biological membranes is often discussed in terms of cholesterol-lipid interactions. Preferential interactions of cholesterol with lipids, varying in headgroup and acyl chain unsaturation, were studied by measuring the partition coefficient for cholesterol in unilamellar vesicles. A novel vesicle-cyclodextrin system was used, which precludes the possibility of cross-contamination between donor-acceptor vesicles or the need to modify one of the vesicle populations. Variation in phospholipid headgroup resulted in cholesterol partitioning in the order of sphingomyelin (SM) > phosphatidylserine > phosphatidylcholine (PC) > phosphatidylenthanolamine (PE), spanning a range of partition DeltaG of -1181 cal/mol to +683 cal/mol for SM and PE, respectively. Among the acyl chains examined, the order of cholesterol partitioning was 18:0(stearic acid),18:1n-9(oleic acid) PC > di18:1n-9PC > di18:1n-12(petroselenic acid) PC > di18:2n-6(linoleic acid) PC > 16:0(palmitic acid),22:6n-3(DHA) PC > di18:3n-3(alpha-linolenic acid) PC > di22:6n-3PC with a range in partition DeltaG of 913 cal/mol. Our results suggest that the large differences observed in cholesterol-lipid interactions contribute to the forces responsible for lateral domain formation in plasma membranes. These differences may also be responsible for the heterogeneous cholesterol distribution in cellular membranes, where cholesterol is highly enriched in plasma membranes and relatively depleted in intracellular membranes.

[23] Purification of rhodopsin by concanavalin A affinity chromatography

Publisher Summary This chapter discusses the purification of rhodopsin by concanavalin a affinity chromatography. Affinity chromatography represents one of the major advances in protein purification methodology. This technique depends on the reversible binding of a ligand to a protein. Rhodopsin has been identified as a mannose-containing glycoprotein and as such has the potential ligand properties for affinity chromatographic purification. The procedure to be described employs the nonionic detergent octyl glucoside. Rhodopsin fractions are pooled and can be concentrated by ultrafiltration using a Diaflo PM 10 or PM 30 filter. Pooled samples are generally concentrated to about 3 mg/ml of rhodopsin, involving a reduction in volume from about 40 ml to 5 ml. This process results in a concentration of octyl glucoside and rhodopsin, the final detergent concentration being in the range of 60 mM to 70 mM; this raises no concern with respect to rhodopsin stability, as rhodopsin is stable in octyl glucoside solutions as high as 300 mM.

Effect of Cholesterol on Molecular Order and Dynamics in Highly Polyunsaturated Phospholipid Bilayers

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.

Manipulation of Cholesterol Levels in Rod Disk Membranes by Methyl-β-cyclodextrin EFFECTS ON RECEPTOR ACTIVATION

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-β-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 ± 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter fv . The level of MII formed showed a positive linear correlation with fv over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.

[91] Fluorescent probe: Diphenylhexatriene

Publisher Summary This chapter discusses the fluorescent probe—diphenylhexatriene. Depolarization of fluorescence has proved to be a very reliable technique with which to characterize the thermotropic and dynamic aspects of the hydrophobic regions of lipid bilayers and lipoproteins. Fluorescence analysis of dynamic systems can be performed either with time-dependent or steady-state type anisotropy measurements. The former approach can yield information concerning the heterogeneity of the lifetime of the fluorophore in the system, allowing in some cases a resolution of fluorophore subpopulations. This technique also allows an evaluation of the hindrance of the probe motion because of the degree of order of the phospholipid acyl side chains. Fluorescence polarization is measured by exciting the fluorophore with monochromatic light through a polarizer whose polarization axis is oriented vertically to the light path and the emission intensity is detected through an analyzer, whose polarization axis is oriented sequentially parallel to and perpendicular to the polarization axis of the exciting light.

Molecular Order and Dynamics in Bilayers Consisting of Highly Polyunsaturated Phospholipids

The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic bilayer structural properties of symmetrically substituted phosphatidylcholines (PCs) with acyl chains containing no, one, four, or six double bonds and mixed-chain phosphatidylcholines with a saturated sn-1 chain and one, four, or six double bonds in the sn-2 chain. Both the Brownian rotational diffusion (BRD) model and the wobble-in-cone model were fit to all differential polarization data, and the descriptions of the data provided by the BRD model were found to be statistically superior. Global analysis of differential polarization data revealed two statistically equivalent solutions. The solution corresponding to a bimodal orientational distribution function, f(theta), was selected based on the effects of temperature on f(theta) and previous measurements on fixed, oriented bilayers. The overall equilibrium acyl chain order in these bilayers was analyzed by comparing the orientational probability distribution for DPH, f(theta) sin theta, with a random orientational distribution. Orientational order decreased and probe dynamics increased in mixed-chain species as the unsaturation of the sn-2 chain was increased. The degree of orientational order dropped dramatically in the dipolyunsaturated species compared with the mixed-chain phosphatidylcholines, which contained a polyunsaturated sn-2 chain. In terms of both orientational order and probe dynamics, the differences between the highly polyunsaturated species and the monounsaturated species were much greater than the differences between the monounsaturated species and a disaturated PC.

Optimization of Receptor-G Protein Coupling by Bilayer Lipid Composition I KINETICS OF RHODOPSIN-TRANSDUCIN BINDING

The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (Gt) as a model system. Metarhodopsin II (MII) and MII-Gt complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-Gt formation to a greater extent than the lifetime of MII. MII-Gt formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 °C and a Gt to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-Gt formed with a lifetime of 0.6 ± 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-Gt complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-Gt formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-Gt formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.