Rod Outer Segment Phospholipids Research Articles

Strong association of unesterified [3H]docosahexaenoic acid and [3H-docosahexaenoyl]phosphatidate to rhodopsin during in vivo labeling of frog retinal rod outer segments.

Docosahexaenoic acid (DHA, 22:6n-3), the most prevalent fatty acid in phospholipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [3H]DHA-lipids to rhodopsin in ROS from frogs (Rana pipiens) after in vitro (4 hrs) and in vivo (1 day and 32 days) labeling. Lipids from lyophilized ROS were sequentially extracted with hexane (neutral lipids), chloroform:methanol (phospholipids) and acidified chloroform:methanol (acidic phospholipids). After in vitro labeling, free [3H]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rhodopsin). In contrast, after in vivo labeling free [3H]DHA was mainly recovered in the acidic solvent extract (89-99%). Of all phospholipids, [3H-DHA]phosphatidic acid (PA) displayed the highest binding to rhodopsin after both in vitro (43% in acidic extract) and in vivo (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.

Dietary fat alters membrane composition in rod outer segments in normal and diabetic rats: Impact on content of very-long-chain ( C ⩾ 24) polyenoic fatty acids

The effect of high n − 3 (5.8%, w/w) vs. a low n − 3 (1.2%, w/w) fatty acids in a diet with a low ratio of polyunsaturated/saturated fatty acids ( P/ S = 0.27) content was investigated to determine the effect of diet on the level of long-and very-long-chain fatty acids ( VLCFA C ⩾ 24) in phospholipids of rod outer segments (ROS) of normal and diabetic rats. After 6 weeks of feeding, diets high in n − 3 fatty acids increased the levels of 22:5( n − 3) and 22:6( n − 3), while decreasing the 22:5( n − 6) level in all major phospholipid classes, n − 6 and n − 3 VLCFA of C24 to C34 with 4, 5 and 6 double bonds were found only in phosphatidylcholine (PC) while other phospholipid classes contained only C24 fatty acids as minor components. The content of VLCFA in PC was approx. 6.7% (w/w) of total fatty acids in the ROS. Feeding a high n − 3 fatty acid diet significantly reduced n − 6 tetraenoic VLCFA in all phospholipids. In the diabetic state, the levels of n − 6 tetraenes and pentaenes in individual phospholipids were different from control animals. This study demonstrates that the VLCFA content of photoreceptor cells reflects the dietary level of n − 3 fatty acids fed. The unique polyenoic n − 6 and n − 3 VLCFA appear to be synthesized from shorter chain precursors which respond to altering the ratio of soln − 6 n − 3 fatty acids fed.

Tunicamycin does not inhibit transport of phosphatidylinositol toXenopus rod outer segments

Tunicamycin inhibits the dolichol pathway for N-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. Adult Xenopus retinas were preincubated in dim red light with 20 micrograms ml-1 of tunicamycin for one hour followed by incubation in the light for 2-6 h with tunicamycin plus either [3H]mannose, [3H]leucine, [2-(3)H]glycerol or [3H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [3H]leucine and [3H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [3H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [3H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [3H]glycerol and [3H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.

Conservation of Docosahexaenoic Acid in Rod Outer Segments of Rat Retina During n‐3 and n‐6 Fatty Acid Deficiency

We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n-9), 20:4(n-6), and 20:5(n-3), respectively. The levels of these 20-carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n-3) with 22:5(n-6), as evidenced by a reduction of the 22:6(n-3)-22:6(n-3) molecular species and an increase in the 22:5(n-6)-22:6(n-3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n-3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n-3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22-carbon polyunsaturated fatty acids from the circulation.

The binding of G-protein to rod outer segment phospholipids at the nitrogen–water interface

In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption-desorption in the monolayer of each main ROS phospholipid. From examination of surface pressure and ellipsometric isotherms at the nitrogen-water interface, we have determined that once incorporated into the monolayer, the G-protein remains associated, independent of surface pressure, thus providing evidence against the hopping model.

Metabolic labeling of normal canine rod outer segment phospholipids in vivo and in vitro

Twenty-four hours after the intravitreal injection of [ 3H]palmitate and [ 14C]docosahexaenoate in dogs, the rod outer segment phospholipids are highly labeled. Palmitate is found predominantly in phosphatidylcholine (PC), with lesser amounts in phosphatidylethanolamine (PE) and very little in either phosphatidylserine (PS) or phosphatidylinositol (PI). Docosahexaenoate most heavily labeled PE followed by PC, with lesser amounts in PS and very little in PI. Two-hour incubations of 3 mm trephine buttons removed from dog retinas produced very similar patterns of labeling with palmitate and docosahexaenoate. In vitro incubation of retina buttons with [ 3H]arachidonate produced heavy labeling of PI, with much less in PC and very little in either PS or PE. [ 3H]Glycerol labeled in PC, PI and PE in descending order but PS almost not at all, [ 3H]Serine labeled PS predominantly, but small amounts were found in PC, PE and PI. The trephine retina buttons can be utilized for multiple-precursor incubations and studies of differential metabolism in retinal regions, particularly when studying scarce tissue from mutant animals or humans with inherited retinal degenerations.

Molar absorptivities of bovine retina rod outer segment phospholipids in n-hexane

The molar absorptivities of the major bovine rod outer segment (ROS) phospholipids and phophatidylcholine 18:1 have been determined at four wavelengths, i.e., 193.5, 196, 200, and 205 nm. The mean standard error is 1.7% at 95% confidence level. The results obtained can therefore be used for the quantitative analysis of bovine ROS phospholipids. Compared with the existing methods, the spectrophotometric determination of these lipids presents the advantages of being rapid, direct, and very sensitive. The importance of stray light in this type of measurement is also discussed.

Ellipsometric studies of rod outer segment phospholipids at the nitrogen-water interface

Ellipsometry is an optical method which uses the principle that the state of polarization of polarized light changes on reflection at an interface. We have constructed a null ellipsometer for the study of the optical properties of monomolecular films of biological interest at the nitrogen-water interface. With this apparatus we have reproduced the known ellipsometric properties of arachidic acid spread on acidic aqueous solution. Then, we have performed surface potential, surface pressure and ellipsometric measurements of PC (18:1) and rod outer segment phospholipids, i.e. PE ros , PC ros and PS ros , at the nitrogen-water interface. Based on literature refractive indices data, results provide information about the thickness of a lipid film in the close-packed region. Pertinent information about molecular organization of the film is deduced from the close relationship observed between surface potential and ellipsometric isotherms. The importance of these results on the discal membrane structure and function is discussed.

92] Analysis of rod outer segment disk membrane phospholipid organization using parinaric acid fluorescent probes

Publisher Summary This chapter considers the application of the parinaric acid (PnA) fluorescent probes to the analysis of rod outer segment membrane phospholipid organization. This analysis is comprised of several parts: (1) the partition of PnA probes between aqueous phase and embranes, (2) the identification of phospholipid phase separation in rod outer segment membrane lipids with PnA probes, (3) the partition of PnA isomers between coexisting fluid and solid phospholipid phases, (4) the reconstruction of the thermal reorganization in rod outer segment membranes based on measurements of model dispersions comprising the individual membrane monolayers, and (5) the effects of Ca 2+ on rod outer segment phospholipids. This chapter concludes that membranes with broad, relatively featureless thermal behavior may well contain a superposition of relatively sharp thermal components. PnA probes or their methyl ester derivatives are readily incorporated into stirred membrane suspensions or phospholipid dispersions at 37 ° when added slowly as microliter aliquots of dilute ethanolic solutions (

44] Determination of molecular species of rod outer segment phospholipids

Publisher Summary This chapter discusses the determination of molecular species of rod outer segment (ROS) phospholipids. The total phospholipid fraction is resolved into individual lipid classes by two-dimensional thin-layer chromatography (TLC). Two studies of ROS molecular species have indicated that they are rather unique because they contain large proportions of dipolyunsaturated species. The molecular species of a phospholipid class is calculated to the best fit estimate by using proportional analysis, algebraic manipulations, and normalization of all the data from the resolved diglyceride acetates (DGAC) molecular classes and their fatty acid composition and distribution. Positional analysis of the phospholipid fatty acids is performed by subjecting each phospholipid to phospholipase A 2 hydrolysis, which specifically releases the acids from the 2-position. Positional analysis of the fatty acids for the total DGAC mixture and for each DGAC molecular class is accomplished with pancreatic lipase hydrolysis. The results of the molecular species determination will be a quantitative chemical description of the major phospholipids found in rod outer segments.

The effect of light stimulation on phospholipids of calf rod outer segments

To determine whether light has an effect on the lipids of calf rod outer segment membranes, total phospholipids, alkenyl phospholipids and phospholipid fatty acids were analysed. Ethanolamine phosphoglycerides, choline phosphoglycerides and serine phosphoglycerides were the major phospholipids of calf rod outer segments. They accounted for 43, 37 and 14% of total lipid phosphorus respectively. As in the whole retina, alkenyl phospholipids were found, but in lower amounts. The choline plasmalogens accounted for 3% of total choline phosphoglycerides, and ethanolamine plasmalogens 7% of total ethanolamine phosphoglycerides. The unsaturated fatty acids made up 50% of total fatty acids. The major fatty acids of the rod outer segment phospholipids were docosahexaenoate (41%), palmitate (13%) and stearate (23%). No significant differences were observed between the outer segments kept in the dark or illuminated. This result could not exclude a more specific action of light on certain polyunsaturated fatty acids, possibly on docosahexaenoate which is a major component of the rod outer segments.

Lipids of ocular tissues: VII. Positional distribution of the fatty acids in the phospholipids of bovine retina rod outer segments

The positional distribution of the fatty acids in the major phospholipids of bovine retina rod outer segments was determined. Phosphatidyl ethanolamine and phosphatidyl serine have mostly saturated acids in the 1-position and docosahexaenoic acid in position 2. These phospholipids contain 94 and 79%, respectively, of polyun-saturated acids in the 2-position. Phosphatidyl choline contains mostly saturated acids in the 1-position, but has significant quantities of palmitic in the 2-position along with docosahexaenoic acid. The levels of docosahexaenoic acid in rod outer segment phospholipids are among the highest yet reported for membrane phospholipids, amounting to 23% in phosphatidyl choline, 39% in phosphatidyl ethanolamine, and 45% in phosphatidyl serine.