Radioactive Lipids Research Articles

Axonal transport of lipids in the rabbit optic system.

Abstract— Axonal transport of lipids was demonstrated in the rabbit optic system using [2‐3H]glycerol and [3‐14C]serine. Following intraocular injection of these precursors, radioactive lipids were detected in the optic tract, superior colliculus and lateral geniculate body over a 31 day period. The bulk of lipid appeared to migrate at a rate equivalent to that of rapidly transported protein which, when combined with a prolonged period of release into the axon, led to a peak of transported radioactivity at 6‐10 days for the 3 tissues. The suggestion of a second peak at 17 days indicated the possibility of a smaller slow component, although another interpretation is suggested. Analysis of individual transported lipids revealed [2‐3H]glycerol to label phosphoglycerides preferentially and [3‐14C]serine to be an effective precursor for sphingolipids and certain of the phosphoglycerides. [3‐14C]Serine labeled axonally transported proteins to an even greater extent than lipids, revealing the same fast and slow components previously shown with other amino acids.

METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1‐14C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half‐lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half‐life for turnover of synaptosomal lipids was 9 days.The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl‐GPC and diacyl‐GPI, since radioactivity of other phosphoglycerides (diacyl‐GPS, diacyl‐GPE and alkenyl‐acyl‐GPE) continued to increase for 12‐24 h. In this fraction, half‐lives of 10‐14 h were obtained for the fast turnover pools of diacyl‐GPC and diacyl‐GPI, and slow turnover pools with half‐lives of 7 days for diacyl‐GPI and 10‐14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl‐GPI declined in a biphasic mode, thus exhibiting half‐lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl‐GPE and diacyl‐GPS in the synaptosomal fractions was observed for a period of 24 h. The half‐lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl‐GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.

The effect of a putative anti-atherosclerotic agent (S 1204) on lipid metabolism in rabbit aorta

The effects of the novel fenfluramine derivative, S 1204 (meta-trifluoromethyl phenyl-1 [β(sulfamyl-3′-chloro-4′-benzoyloxyethyl)]amino-2-propane) were studied on lipid metabolism in rabbit aorta and other tissues. Pretreatment of rabbits with S 1204 (50 mg/kg orally) for 10 days strongly inhibited the aortic incorporation of an intravenous 20 μCi tracer-dose of [4- 14C]-cholesterol given 24 h earlier. However, incorporation of such cholesterol radioactivity into the liver, intestine, lung or plasma was not affected, in comparison with control animals. One hour after i.v. injection of a 100 μCi tracer-dose of [2- 14C]acetate, S 1204 significantly reduced the radioactivity of total lipids in the aorta and liver. Such inhibition of acetate incorporation into arterial lipids was observed with all lipid fractions (that is, free fatty acids, esterified cholesterol, especially phospholipids, glycerides and free cholesterol). Incorporation of acetate into intestinal triglyceride fractions was also significantly decreased. The results indicate that S 1204 may have anti-atherogenic properties, which could be valuable in the clinical treatment of atherosclerosis.

Evidence for rate-limiting steps in sterol synthesis beyond 3-hydroxy-3-methylglutaryl-coenzyme a reductase in human leukocytes

When human blood leukocytes are incubated with [2- 14C] acetate only about 32% of the nonsaponifiable lipid radioactivity is recovered in digitonin-precipitable material. Using thin-layer chromatography and gas-liquid radiochromatography, we have determined that most of the label from [2- 14C] acetate in the nonsaponifiable fractions is in lanosterol, squalene and an unidentified sterol. Only 11% of the acetate radioactivity is contained in cholesterol. This distribution does not change when cholesterol synthesis is depressed by the addition of lipoproteins to the medium. These findings are in marked contrast to studies with liver, where most of the nonsaponifiable radioactivity derived from acetate is recovered in digitonin-precipitable sterols. Furthermore, they suggest that rate-limiting steps beyond the 3-hydroxy-3-methylglutaryl coenzyme A reductase reaction exist in the sterol synthesis pathway of human leukocytes.

The synthesis of choline phosphoglycerides in the giant fibre system of the squid.

Abstract—The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion‐nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP‐choline in the intact ganglion‐nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base‐exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations.Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.

Lipid transport mechanism in the prawn.

The distribution of radioactive lipid classes in the tissues of prawn, Penaeus japonicus, was investigated after oral administration of linolenic acid-1-14C in order to clarify the mechanism of lipid transport in crustaceans. Also, the movement of linolenic acid to the serous membrane site across the hin-dgut was examined by using the everted hind-gut of prawn. Dietary free fatty acid was presumed to be mainly absorbed at the region of the hepatopancreas and mid-gut rather than at the hind-gut. The absorbed linolenic acid was resynthesized to phospholipids (PL) in the hepatopancreas and hind-gut, and then released into the hemolymph as a form of PL. From this data, PL were conceived to be the most important mediators of lipid mobilization through the hemolymph in the prawn, P. japonicus.

Digestion and absorption of lecithin in larvae of the cabbage butterfly, Pieris brassicae

1. 1. Dietary lecithin [phosphatidyl ( N-methyl- 14C) choline] is converted to the surface-active lysolecithin and to water-soluble metabolites in the intestinal lumen of the butterfly, Pieris brassicae. 2. 2. In the midgut cells radioactivity was found in lecithin, lysolecithin and sphingomyelin. 3. 3. In the haemolymph over 90% of the radioactivity was water-soluble after 6hr, but only 32% was water-soluble after 48 hr of feeding. Labeled lipids in the haemolymph were lecithin ( ca. 90% of lipid radioactivity) and sphingomyelin. 4. 4. Based on an assay of lipid phosphorus, dietary lecithin was found to be utilized by the larvae almost completely. The titre and specific activity of midgut lysolecithin suggest a role for this lipid in absorption.

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.

Autoregulation of phospholipid N-methylation by the membrane phosphatidylethanolamine content

1. Introduction The step-wise methylation of phosphatidylethanol- amine is catalyzed by membrane-bound enzyme(s), with high activity in liver endoplasmic reticulum [l] . The rate of methylation is influenced by the concentra- tion of one of the reactants, Ado-Met (S-adenosyl- methionine) in experiments with subcellular fractions [l] and on the availability of methionine in isolated hepatocytes [2] and in intact rats [3]. Nothing is known about the influence of the other substrate, phosphatidylethanolamine, since phosphatidyl- ethanolamine added to microsomal systems does not stimulate methylation, in contrast to phosphatidyl-IV- methylethanolamine and phosphatidyl-NN-dimethyl- ethanolamine [ 1,4] . We have recently shown that the amount of phos- phatidylethanolamine in hepatocytes can be manipu- lated by maintaining them in primary culture in the presence and absence of ethanolamine [5]. This report deals with the several-fold variation in phospholipid methylation in such cells. 2. Materials aud methods Livers were perfused with collagenase and the hepatocytes were isolated and plated in medium L-l 5 on Petri dishes as in [5]. The first change of medium was after l-2 h and then the medium was changed once a day. After incubation with different agents, the medium was aspirated and replaced by new medium and [‘4C]methionine (0.5 mM, which is the concentration in medium L 15). After incubation for l-2 h at 37”C, the medium was aspirated and lipids were extracted from the cells [5]. Total phospholipid content in each lipid extract was determined [6] and the incorporation of r4C from [methyl-14C]methionine was expressed as nmol/lipid P. Phosphatidylethanol- amine was determined with trinitrobenzenesulphonic acid [7]. Alternatively the cells in the Petri dish were washed with 2 ml of cold NaCl(9 mg/ml) containing 1 mM Tris-HCl, pH 7.4. The cells were then transferred with four 0.5 ml portions of the same solution to centrifuge tubes and were disrupted by sonication for 30 s. The activity of phosphatidylethanolamine methyl- transferase (EC 2.1 .l .17) was measured with a scaled- down modification of the method in [4]. The assay mixture contained 0.05 M Tris-HCl (pH 8.2) and cell homogenate (0.5 mg protein) in total vol. 0.5 ml. The reaction was started by the addition of 0.04 mM [methyl-3H]Ado-Met (Radiochemical Centre, Amersharn, Bucks) and the samples were incubated by shaking for 30 min or 60 min. The reaction was interrupted by the addition of 3 ml chloroform/ methanol (1: 1). After 30 min 1 ml water and 1.5 ml chloroform were added and then the chloroform phase was washed twice with 3 ml methanol/water (1: 1, saturated with chloroform). Total lipid radioactivity was routinely measured with liquid scintillation counting. Occasional checks by thin-layer chromato- graphy [5] showed that 90% lipid radioactivity was always in phosphatidylcholine.

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.

Effect of metformin on lipid metabolism in the rabbit aortic wall

The aim of the present study was to investigate whether metformin was capable of altering aortic lipid metabolism. Pretreatment of rabbits for 8 days with 120 mg/kg per os metformin reduced by 50–70% the incorporation of a 20 μCi tracer dose of [4- 14C]cholesterol (given orally 24 h before) into various segments of aorta, plasma, liver, intestine and lung, as compared with control animals. However, as intestinal absorption of cholesterol was also diminished in the same proportion, it was then decided to inject the labelled cholesterol directly into the blood. Under these conditions, metformin induced the same reduction in [4- 14C]cholesterol specific activity in the aorta, but not in other tissues. Three hours after intravenous injection of a 200 μCi tracer dose of [2- 14C]-acetate, metformin strongly diminished the radioactivity of total lipids of the aorta, both in fed-rabbits and in rabbits on a 24 h fast, independently of the plasma radioactivity level. The inhibition of acetate incorporation into arterial lipids was observed in all lipid fractions (i.e. free and esterified cholesterol, free fatty acids, phospholipids and especially triglycerides) and the effect persisted unaltered over periods of increasing length after the injection of precursor (1/4, 1/2, 1, 3, 5, 8, 12 h). Metformin also significantly inhibited lipid biosynthesis in the liver and intestine. These properties, added to others previously described, can to a large extent explain the preventive effect of metformin on experimental atherosclerosis.

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes.

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.

Subcellular distribution of [1- 14C]palmitate and [1- 14C]oleate incorporated into lipuids in the perfused rat heart: A comparison under isothermal and hypothermic conditions

Isolated rat hearts have been perfused with [1- 14C]palmitate and [1- 14C]oleate for varying periods of time followed by unlabelled perfusion for varying periods of time. The degree of incorporation of the label into the lipids of mitochondrial, microsomal and cytoplasmic fractions has been studied. At the same time lipid separation by thin layer chromatography has given an indication of rates and location of esterification to triglyceride. The results have been analyzed in the light of the previously defined limitations of the methods of cell fractionation. The accumulation of lipid radioactivity reached a plateau in the cytoplasmic and mitochondrial fractions at 60 s. Radioactivity continued to increase in the microsomal fraction due to the progressive formation of triglyceride. The formation of triglyceride was dependent on temperature. The uptake of palmitate into lipid was greater than that of oleate.

What is the origin of the lipids of potato tuber plasmalemma?

Plasmalemma isolated from potato tuber cells was prepared according to a flotation method in a sucrose density gradient. When incubated in vitro with various radioactive lipid precursors, the plasmalemma fragments were unable to synthesize any lipid. When labelled lipid precursors ([ 14C]acetate of [ 32P]phosphate) were furnished to potato slices, radioactive fatty acids or phospholipids were integrated into plasmalemma lipids. When incubated in a suspension of [ 32P]liposomes, plasmalemma fragments accepted labelled lipids from the suspension; this exchange process was markedly stimulated by the phospholipid exchange protein prepared from the potato cytoplasmic supernatant. These results suggest that potato tuber plasmalemma is dependent on other cellular fractions for lipid synthesis and exchange.

Aspects of lipid synthesis, hydrolysis, and transport studied in selected Amazon fish

Comparative lipogenic activities offish tissue, lipolytic activity of mesenteric fat and mode of intestinal lipid release into blood were investigated in a variety of Amazon fish. Small catfish were injected intramuscularly with [1-l4C]acetate, killed at intervals, and the lipid radioactivity of 11 separate tissues determined. In 6- and 18-h fish, the heart, eyes, dark muscle, and white muscle synthesized negligible lipid relative to the other tissues. Acetone powders of Triportheus sp, mesenteric fat contained high amounts of triglyceride lipase activity (120 nmol fatty acid release-d/min per milligram protein). The activity exhibited a pH optimum of 8.0 and was not activated by albumin, bile salts, or divalent salts nor inhibited by 1 M NaCl. Characteristics of the observed activity are identical with those of mammalian pancreatic lipase. Hoplias malabaricus were fed [1-14C]oleic acid and a chronic indwelling catheter was placed in the dorsal aorta for blood sampling. Based on the distribution of radioactivity among blood lipid classes, it is suggested that dietary lipid enters fish circulatory systems both as free fatty acid and as lipoproteins.

Distribution of radioactivity in myelin lipids following subcutaneous injection of [ 14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).

NOREPINEPHRINE‐STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE OF RABBIT IRIS SMOOTH MUSCLE: EFFECTS OF SURGICAL SYMPATHETIC DENERVATION AND IN VIVO ELECTRICAL STIMULATION OF THE SYMPATHETIC NERVE OF THE EYE

Abstract— Paired iris smooth muscles from rabbits were prelabelled either in vitro by incubation for 30 min at 37°C in an iso‐osmotic salt medium containing glucose, inositol, cytidine and 32Pi, or in vivo by administration of the isotope intracamerally into each eye 1 h before death. One of the pair was then incubated at 37°C for 10 min in an unlabelled medium containing 10 mm of 2‐deoxyglucose and the other was incubated in the presence of norepinephrine (NE) or other adrenergic agents. Triphosphoinositide (TPI) was found to contain more 32P than any other phospholipid (almost 39% of total lipid radioactivity) in both the in vitro and in vivo experiments. NE (50 μm) increased the loss of 32P from TPI (the TPI effect') by 28–30% in the 32P‐labelled muscle. The TPI effect was accompanied by a significant increase in 32P labelling of phosphatidic acid (PA) and phosphatidylinositol (PI) but not phosphatidylcoholine. In this tissue the TPI effect was found to be mediated through α‐adrenergic receptors. At 14 days after surgical sympathetic denervation, incorporation of 32P into phospholipids of the denervated muscle increased by an average of 6% over that of the normal muscle. The increase in TPI, PI and PA was 7%, 4% and 9% of that of the control respectively. There was little change in phospholipid content of the denervated muscle. The increase in sensitivity to NE (12.5 μm) caused by denervation produced about 18% increase in the TPI effect and a 25% increase in the 32P labelling of PA, but not PI. In view of our previous findings on the requirement of the TPI effect for Ca2+, this observation could suggest that an increase in Ca2+ influx, following the interaction between the neurotransmitter and its receptor could stimulate TPI‐phosphodiesterase, thus leading to increased PA via increased diglyceride. This denervation‐induced supersensitivity to NE appears to be postsynaptic in nature. 32Pi was injected intracamerally into each eye 1 h before electrical stimulation of one of the sympathetic trunks. After stimulation for 30 min there was a significant loss of 32P from TPI and a significant increase in the labelling of PI and PA of the stimulated muscle. It is concluded that TPI and its enzymes could play an important role in neurotransmission at the neuromuscular junction of smooth muscle.

Variation in lipid synthesis from acetate during the molting cycle of prawn.

After the injection of sodium acetate-1-14C into some prawns, Penaeus japonicus, the de novo lipid synthesis and the distribution of radioactive lipids in tissues such as the muscle, integument, eyestalk, hepatopancreas, and remains were investigated in association with the molting stages. The incorporation of acetate-14C into the lipids was high at stage C1-C2 and low at stages D1' and D1''-D2'''. Throughout the molting cycle, nearly half of the radioactive lipid was distributed in the remains. In the muscle, eyestalk, integument, and remains, radioactive lipids were present mainly as polar lipids in every stage. In the case of the hepatopancreas, rather large amounts of neutral lipids were present besides the polar lipids, and the ratio of polar to neutral lipids reached a minimum at stage D1'.

Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro.

Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.

Interactions of Phospholipid Vesicles with Murine Lymphocytes

The interactions of mouse thymocytes with unilamellar phospholipid vesicles comprised of dimyristoyl lecithin (DML), dipalmitoyl (DPL), dioleoyl lecithin (DOL), and egg yolk lecithin (EYL) were examined in vitro. In cells treated with [3H]DML or [3H]DPL vesicles, electron croscope (EM) autoradiographic analysis showed most of the radioactive lipids to be confined to the cell surface. Transmission EM studies showed the presence of intact vesicles (DPL) and collapsed or ruptured vesicle fragments (DML) adsorbed to the surfaces of treated cells. In cells treated with DPL vesicles containing a water-soluble dye (6-carboxyfluorescein; 6-CF), most of the fluorescent vesicles were localized at the periphery of the treated cells. Furthermore, substantial fractions of the cell-associated DPL and DML could be released by a mild trypsinization without damaging the cells. These results suggest that the uptake of DML and DPL is primarily due to vesicle-cell adsorption. Such an adsorption process appears to be enhanced at or below the thermotropic-phase transition temperature of the vesicle lipid. Under certain conditions these adherent vesicles also formed patches or caps on the cell surface. In cells treated with DOL or EYL vesicles, transmission EM and EM autoradiography showed relatively little exogenous vesicle lipid located at the cell surface. Thymocytes incubated (37 degrees C) with [14C] EYL vesicles containing a trapped marker, [3H]inulin, incorporated both isotopes at identical rates. In separate experiments it was found that this marker was located inside the treated cells. Thymocytes treated with DOL vesicles containing 6-CF exhibited a uniform and diffuse distribution of dye in the internal volume of the cells. Little cell-associated EYL or DOL could be released by trypsinization. Evidence against endocytosis of intact vesicles as a major pathway of vesicle uptake is also presented. These observations, coupled with the demonstration of vesicle-cell lipid exchange as a minor component of vesicle uptake suggest that incorporation of EYL and DOL vesicles by thymocytes is primarily by vesicle-cell fusion.