[105] Renewal of lipids in rod outer segments

800

[ 105]

BIOGENESIS

[105] Renewal

of Lipids

in Rod

Outer

Segments

B y ROBERT E. ANDERSON

Introduction The lipid and protein c o m p o n e n t s of retinal rod outer segments (ROS) are constantly being renewed. The integral protein rhodopsin is synthesized in the inner segment and, after glycosylation, is transported to the base of the outer segment where it is incorporated into the basal infoldings of the plasma membrane. 1-3 These infoldings eventually pinch off to form free-floating disks within which rhodopsin molecules remain until the disks reach the tip o f the ROS and are shed and phagocytized by the retinal pigment epithelium, a ROS lipids also are synthesized in the inner segment and are probably cotransported with newly synthesized opsin to the base o f the growing outer segment. H o w e v e r , once incorporated into a p h o t o r e c e p t o r disk, the lipid, unlike the protein, freely diffuses between the disks of the rod outer segment. 5-s Over the past several years, we have examined in some detail the synthesis and renewal of ROS lipids. 5-s In this chapter, I will describe the methodology used in these in vivo studies. Animals a n d E n v i r o n m e n t a l C o n d i t i o n s The leopard frog ( R a n a p i p i e n s ) is a good choice of experimental animal for in vivo studies because (1) it can be easily maintained in captivity; (2) it is economical with regard to price, animal maintenance, food, and radioisotope requirements; and (3) it gives a high yield of purified ROS (e.g., about 200/zg of ROS phospholipid can be obtained from one 25 g frog, c o m p a r e d to only about 40/xg of ROS lipid from a 300 g rat). One disadvantage is that their availability is seasonal, but this can be overcome by maintaining a large stock of frogs. Exp. Eye Res. 18, 215 (1974). Invest. Ophthalmol. 15, 7 0 0 (1974). Recept. Recognition, Ser. A 6, 109 (1978). 4 M. O. Hall, D. Bok, and A. D. E. Bacharach, J. Mol. Biol. 45, 397 (1969). 5 R. E. Anderson, P. A. Kelleher, M. B. Maude, and T. M. Maida, Neurochem. Int. 1, 29 1 R. W . Y o u n g , R. W . Y o u n g , 3 p. j. O'Brien,

(1980). 8 R. E. Anderson, M. B. Maude, and P. A. Kelleher, Biochim. Biophys. Acta 620, 236 (1980). 7 R. E. Anderson, M. B. Maude, P. A. Kelleher, T. M. Maida, and S. F. Basinger, Biochim. Biophys. Acta 620, 212 (1980). s R. E. Anderson, P. A. Kelleher, and M. B. Maude,Biochim. Biophys. Acta 620, 227 (1980). METHODS 1N ENZYMOLOGY, VOL. 81

Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7

[105]

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Light cycles, light intensity, and temperature should be stringently controlled. This is best achieved in an environmental chamber such as the modified refrigerators that are commercially available. In our laboratory, frogs are kept in clear plastic cages covered with window screen on a cycle of 14 hr light: 10 hr dark under fluorescent illumination of not more than 350 lumens/m 2. The floor of the cages is covered with about 11.5 cm tapwater (changed daily), and the cages are placed on a slant so that a portion of the cage floor remains dry. The incubator temperature may be manipulated to control metabolic rates, but it should be noted that the body temperature of frogs will not be that of the incubator and should be determined with a stomach probe. All animals are maintained in this rigidly controlled environment for at least one week prior to injection of radioisotopes. Radioisotopes: Dosage and Injection Scheme For in vivo experiments all radioisotopes should be of the highest specific activity available. Tritiated serine, ethanolamine, or choline are injected into the dorsal lymph sac of flogs at a dosage of 1.25/xCi/g body weight. [3H]Glycerol is injected at a dosage of 7-10 tzCi/g. Since significant labeling of retinal fatty acids is obtained from [1,3-3H]glycerol, [23H]glycerol is preferred for studies of glycerolipid turnover in the ROS. 32PO4 (5/zCi/g) is utilized for short-term studies while 33PO4 (10/zCi/g) is preferred for longer term studies. Tritiated inositol is injected at a dose of 2.5/zCi/g. The injection volume should be adjusted so that a 25 g frog receives 0.1 ml. Some of the isotopes are shipped in ethanol; however, this toxic compound does not affect the animals if diluted to 25% or less of the injection volume. All dilutions for injections are made with lactated Ringer's or physiological saline.

Isolation of Retinas At least two groups of four to five frogs each should be sampled at each time point and worked up independently. Animals are dark-adapted for at least 2 hr, decapitated, pithed, and enucleated. The eye is hemisected at the ora serrata and the retina peeled as cleanly as possible from the pigment epithelium-choroid. The retina is placed on an inverted petri dish in a puddle of Tris-Mg 2+ buffer (see below), and any adhering pigment epithelium is removed with a pair of fine forceps. Several other tissues should also be sampled at this time to serve as a comparison for lipid metabolism in the retina. The most frequently taken are brain, liver, and red blood cells.

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BIOGENESIS

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Preparation of Rod Outer Segment ROS are prepared by our modification of the procedure of Papermaster and Dreyer2Retinas are homogenized in 2 ml of 1.170 g/ml sucrose in 10 mM Tris buffer (pH 7.35) containing 2 mM MgC12. Homogenization is achieved by five strokes of a Teflon pestle with a serrated tip in a glass homogenization tube. Clearance between pestle and tube is 0.100.15 mm (Arthur Thomas, catalog no. 3431-E15). This homogenate is transferred to an 18 ml cellulose nitrate centrifuge tube, and the homogenization tube rinsed with 2 ml 1.170 g/ml sucrose, which is also added to the centrifuge tube. The 4 ml of 1.17 g/ml sucrose are overlaid sequentially with 5 ml of 1.150 g/ml sucrose in buffer, 4 ml of 1.135 g/ml, and 5 ml of 1.110 g/ml. Membranes are separated by centrifugation in a swinging bucket rotor (Beckman SW27.1 or Sorvall AH-627) at 82,000 g for 60 min. All procedures to this point are carried out under dim red light. The fluffy pink layer at the 1.110/1.135 interface is removed in the light and diluted to 12 ml with Tris-Mg 2+ buffer, and membranes are pelleted at 18,800 g for 15 min. The pellet is washed two times with buffer and centrifuged at 18,800 g for 15 min. The remaining solution from the discontinuous sucrose centrifugation (approximately 15 ml) is mixed with 15 ml of buffer and centrifuged for 15 min at 18,800 g. This rest-of-retina pellet contains some ROS material, mitochondria, and other heavy membrane fractions. The supernatant is centrifuged at 96,000 g for 90 min to obtain a microsomal pellet. Criteria for Purity of ROS Over 75% of the rhodopsin is recovered at the 1.110/1.135 g/ml interface. The A278nm/A498n m should be less than 3; polyacrylamide gel disk electrophoresis should reveal a protein of molecular weight of around 36,000 that accounts for 90% of the membrane protein; there should be only rare mitochondria or other identifiable organelles in electron microscopic examinations of the membrane preparation; and there should be no evidence of plasmalogens. The latter are present in large amounts in phosphatidylethanolamine (PE) from whole retina, but are not present in PE in the ROS. Extraction of ROS Lipids Wet membranes are homogenized in at least 20 vol of chloroform/methanol (2:1, v/v) in a glass-on-glass homogenizer. Two-tenths 9 D. Papermaster and W. J. Dreyer, Biochemistry 13, 2438 (1974).

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volumes of 0.9% of sodium chloride are added, the tube is shaken, and the extract is separated into two clear phases by low-speed centrifugation. The bottom chloroform layer containing the lipids is transferred to a glass screw-top tube and the solvent evaporated under a stream of dry nitrogen. Lipids are made up to a known volume in chloroform, flushed with nitrogen, sealed with a Teflon-lined top, and stored in a freezer. Another procedure is described in our chapter on molecular species analyses. TM Either can be used with equal success. These procedures are sufficient for the extraction of all ROS lipids except the polyphosphoinositides. In those instances where isolation of this class of lipids is desired, the extracting solvent is chloroform/methanol/12 N HCI (100:100:6, v/v/v). The extraction mixture is washed with 0.2 vol of 1 N HC1, separated into two clear phases by centrifugation, and the lower phase washed three times with 2/3 vol synthetic upper phase. The lower chloroform layer contains the ROS lipids. This latter procedure should not be used for routine lipid extraction because the acid hydrolyzes plasmalogens to fatty aldehydes and lysophospholipids. Thin-Layer Chromatographic Separation of Retinal Lipids Because of the small amounts of lipids usually present in retinal extracts, in those instances where both neutral lipids and phospholipids will contain radioactivity, both lipid classes should be separated on the same thin-layer plate. The adsorbent of choice is silica gel HR spread 0.25 mm thick on glass plates, to which about 150-250/.~g of lipid is spotted in the bottom left corner about 1 in. in from either side. The solvent system for the first dimension is usually hexane/ether/glacial acetic acid (20:80: 1, v/v/v), which resolves mono-, di-, and triglycerides. (Other combinations of the solvents may be used to resolve other types of neutral lipids.) Once removed from the developing tank, the origin is covered with a glass plate, the exposed portion of the plate is sprayed lightly with 2',7'-dichlorofluorescein/methanol/water (0.05 : 75 : 25, w/v/v), and the neutral lipids are visualized under ultraviolet light. Areas corresponding to neutral lipids are scraped from the TLC plate directly into counting vials or saved for determination of lipid mass. The plate is rotated counterclockwise 90 °, and the phospholipids are then separated by conventional twodimensional TLC using a solvent system of chloroform/methanol/glacial acetic acid/0.9% sodium chloride (100:50: 16: 8, by vol) for the first direction and the same solvents in the proportions 100 : 15 : 16:4 for the second dimension. Individual phospholipids are visualized and identified on the chromatoplate following staining in an iodine chamber. lo R. D. Wiegandand R. E. Anderson, this volume,Article [44].

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BIOGENESIS

[105]

Quantitation of Neutral Lipids and Phospholipids Silica gel HR containing individual lipid classes is scraped into a glass tube and digested with perchloric acid, and lipid phosphorus is determined by the procedure of Rouser et a1.11,12 Neutral lipid mass may be quantitated by the high-temperature gas-liquid chromatographic procedure detailed elsewhere in this volume for the study of molecular species of ROS phospholipids. 1°" Although very accurate, this procedure is lengthy and may not be applicable to a large volume of samples. An alternative procedure is to relate the mass of individual neutral lipids in frog ROS extracts to the mass of a phospholipid, such as phosphatidylcholine. Assuming that these proportions will always be the same, it is then possible to estimate the mass of neutral lipids on each TLC plate from the mass of phosphatidylcholine determined by phosphorus assay. Determination of Specific Radioactivity Aliquots of the solution used for phosphorus assay are counted in a liquid scintillation counter, and the specific radioactivity is determined by dividing the DPM by the tzmoles of lipid phosphorus. Recovery of tritium from the chromatoplate following perchloric acid digestion is greater than 90%. However, lipids labeled with the 14C will lose all of their label as 14C02 .

Turnover of Precursor Pools in the Retina To obtain a valid estimate of lipid turnover rates, the labeled precursor itself must be rapidly catabolized so that the tissue is provided with only a pulse of radioactivity. Of the precursors we tested, only glycerol and serine gave true pulses. 5-7 The precursor pools of PO4, inositol, ethanolamine, and choline have a slow turnover in the retina. The turnover rates of precursor pools of all radioisotopes used for lipid synthesis should be tested, and this can be done quite simply by measuring the water-soluble radioactivity in the whole retina and relating the activity to some common denominator such as water-soluble phosphorus. P r e c u r s o r - P r o d u c t Relationships Some estimate of precursor-product relationships between organelles can be made by comparing temporal patterns of specific radioactivities for al G. Rouser, A. N. Siakotos, and S. Fleischer,Lipids 1, 85 (1965). 12G. Rouser, S. Fleischer,and A. Yamamoto,Lipids 5, 494 (1970).

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individual phospholipids. However, one must be cautious in drawing firm conclusions since the only organelle that can be isolated from a known cell-of-origin is the ROS. All of the others are derived from every retinal cell. Phospholipid Interconversions Regardless of whether a general (i.e., glycerol) or a specific (i.e., ethanolamine) lipid precursor is utilized, all lipid classes should be examined for radioactivity so that any metabolic interconversions can be identified. Calculation of T u r n o v e r T i m e of ROS Integral Proteins The turnover time of ROS integral proteins is most accurately determined by measuring the temporal displacement of a band of radioactive protein using standard autoradiographic techniques. 13 Calculation of Half-Life of R O S Lipids This calculation is made by applying linear regression analysis to the log of the specific radioactivity of individual phospholipids vs. time, taking the time of maximum specific radioactivity as the first data point. The half-life thus determined will be a maximum value, since these calculations do not take into account any radioactive lipid incorporated into the rod outer segment after maximum specific activity has been achieved. Relationship b e t w e e n T u r n o v e r T i m e s of R O S Lipid a n d Protein Once incorporated into an ROS disk, integral proteins remain there with that disk until it is shed and phagocytized by the retinal pigment epithelium. 4 As a consequence, the specific activity of ROS integral proteins remains constant as long as the protein is present in the ROS. Conversely, the lipids of ROS turn over in an exponential manner, indicating a diffuse labeling of the R O S ) -s One question to be addressed is whether the lipids and proteins are turning over at the same rate. If it is assumed that they are, then it is possible to determine mathematically the percent of the maximum specific radioactivity of a given phospholipid that should be present after one complete turnover of ROS integral proteins. Assuming that the lipid is lost only through the shedding of tips of ROS, the equation is: % remaining specific radioactivity = 100(1 - X ) Y, where X is the frac~ J. G. Hollyfield,Invest. Ophthalmol. Visual Sci. 18, 977 (1979).

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BIOGENESIS

[106]

tion of an ROS lost with each shedding event and Y is the number of shedding events (X and Y are reciprocals). For example, if frog ROS turnover is 40 days, and since about one-fourth of the ROS shed a disk each day, 14 ten shedding events should occur during this period, and each event should result in loss of 0.1 of the volume of the ROS. Substituting 0.1 and 10 forX and Y, respectively, we calculate that 35% of the maximum radioactivity should be present in phospholipids after one complete turnover of the membranes. Any value less than this indicates that the lipid is turning over more rapidly than the protein, while values greater than 35% indicate that the lipid is turning over more slowly than the integral proteins. In our studies with frogs, we observed that the phospholipid is turning over faster than could be explained by the loss only through the orderly shedding of photoreceptor tips) -s Acknowledgments T h e contributions of Dr. Scott Basinger, M a u r e e n M a u d e , Paula Kelleher, T o m Maida, Lee Plattenburg, and C o n s t a n c e R o e d e r - G o r d o n to the s u c c e s s of t h e s e e x p e r i m e n t s is appreciated. T h e s e studies were s u p p o r t e d in part by grants EY 00871 and EY 07001 from the U S P H S , and grants from the Retina R e s e a r c h Foundation ( H o u s t o n , Texas), B r o w n Foundation (Houston), and R e s e a r c h to P r e v e n t Blindness, Inc.

14 S. Basinger, R. Hoffman, and M. M a t t h e s , Science 194, 1074 (1976).

[106] Fatty Acid Composition and Pairing in Phospholipids of Rod Outer Segments

By GEORGE P. MILJANICH and EDWARD A. DRATZ In common with the phospholipids of most other biological membranes, the phospholipids of the rod outer segment (ROS) disk membrane possess a large measure of molecular heterogeneity. First, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin, and very small amounts of lysophosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylserine have been identified as components of ROS phospholipids. PE, PC, and PS comprise over 95% of the total. Second, each phospholipid class contains a variety of fatty acids, which, in principle, can be paired within each phospholipid molecule (containing two fatty acids) to give a large number of molecular species. The major phospholipids of the ROS disk membrane have now been subfractionated by a simple TLC method METHODS IN ENZYMOLOGY, VOL. 81

Copyright © 1982by AcademicPress, Inc. All rights of reproduction in arty form reserved. ISBN 0-12-181981-7