812

Platelet-Activating Factor Regulates Phospholipid Metabolism in Human Neutrophils Jen-sie Tou Department of Biochemistry, Tulane UniversitySchool of Medicine, N e w Orleans, LA 70112.

This study extended the earlier finding that plateleta c t i v a t i n g factor (1-O-alkyl-2-acetyl-sn-glycero-3phosphocholine} promotes arachidonic acid incorporation into neutrophil phosphatidylinositol {PI) and phosphatidylcholine (PC). In th~ present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10 -7 M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1-10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in ,PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC: These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF. Lipids 24, 812-817 {1989). Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-snglycero-3-phosphocholine) is synthesized in various tissues and stimulated blood cells including neutrophils (1}. It has been shown that the major portion of PAF synthesized by stimulated neutrophils is either released into the extracellular medium {2-5} or remains membrane bound (6,7). High affinity binding of PAF receptors by exogenous PAF having been identified on neutrophil cell membranes (8,9), PAF release into the extracellular medium could occur in vivo. Investigations on the biological effects of PAF on neutrophil metabolism were performed largely in the absence of extracellular fatty acids. However, the concentrations of free fatty acids in the plasma have been estimated to be about 0.5 geq/ml in the basal state {10}, and neutrophils have been shown to be capable of incorporating free f a t t y acids bound to albumin into Abbreviations: DBSS, Dulbecco's balanced salt solution; 5HETE, 5-hydroxyeicosatetraenoic;LTB4, leukotriene B4; MOPS, morpholinopropanesulfonic acid; PAF, platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine}; PBS, phosphate buffered saline; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TLC, thin-layer chromatography. LIPIDS,Vol. 24, No. 9 (1989)

complex lipids {11}. For this reason, it appears to be important to examine the effect of PAF on the uptake and incorporation of extracellular f a t t y acids into phospholipids by neutrophils. It was demonstrated earlier {12) that PAF promotes the incorporation of arachidonic acid into PI and PC by human neutrophils. However, it has not been established whether the increased phospholipid acylation by exogenous fatty acids is secondary to increased fatty acid uptake by PAFstimulated neutrophils. The present study demonstrated that PAF stimulates the uptake of not only arachidonic acid but also of linoleic and palmitic acid by human neutrophils. It also demonstrated that PAF stimulates the incorporation of all three fatty acids into phosphatidylinositol {PI} and both diacyl and alkylacyl phosphatidylcholine {PC}. MATERIALS AND METHODS

Preparation of human neutrophils. Human blood was obtained from healthy donors who had received no medication in the previous two weeks. Each 33 ml portion of venous blood was mixed with 333 units of sodium heparin {Sigma Chemical Co., St. Louis, MO} in 1 ml of 0.9% NaC1 and 5 ml of 5% dextran T500 {Pharmacia LKB, Piscataway, N J} in 0.9% NaC1. The mixture was allowed to sediment at room temperature for 30 min. A leukocyte-rich plasma was removed and centrifuged at 250 • g for 10 min at 20~ The cell pellet obtained from each 100 ml blood was washed once with 20 ml of 0.9% NaC1 and resuspended in 10 ml of the same solution. Contaminated erythrocytes were lysed by adding 30 ml of ice-cold water to each 10 ml of cell suspension. After inversion of the tube for 15 sec, the isotonicity was restored by adding 10 ml of ice-cold 3.4% NaC1 to the tube. After centrifuging the mixture at 250 • g for five minutes at 20~ the cell pellet from 100 ml blood was resuspended in 12 ml Dulbecco's phosphate buffered saline {PBS) {without Ca 2+ and Mg2+, GIBCO, Grand Island, NY). Each 6 ml cell suspensions were layered on 5 ml Lymphocyte Separation Medium {Litton Bionetics, Charleston, SC} in a 15 ml Falcon tube and centrifuged at 400 X g for 20 min at 20~ The resulting neutrophil pellet was washed twice each with 20 ml PBS and once with 20 ml Dulbecco's balanced salt solution (DBSS) {13) containing 5 mM glucose. It was finally resuspended in DBSS containing 5 mM glucose at a concentration of 20 • 106 cells/ml. Cell counts were made in a hemocytometer, and cell viability was measured by trypan blue exclusion. Cell preparations contained more than 95% neutrophils. Incubation of cells. Each radiolabeled fatty acid (New England Nuclear Corp., Boston, MA} was suspended in 0.9% NaC1 containing fatty acid-free bovine serum albumin (4 mglml) (Sigma} at a concentration of 38-43 ~M. PAF {Avanti Polar Lipids, Birmingham, AL} was suspended in f a t t y acid-free bovine serum

813

PAF AND PHOSPHOLIPID ACYLATION albumin (2.5 mg/ml 0.9% NaC1) at a concentration of 20 t~M. Each incubation tube in a final volume of 2 ml contained 0.97 ~M (0.1 ~Ci} [1-14C]arachldonic acid (52.0 Ci/mol) or 1.14 ~M (0.125 ~Ci) [1-,4C]linoleic acid (59.0 Ci/mol} or 0.95 uM (0.1 ~Ci} [1-~4C]palmitic acid (56.0 Ci/mol), 10 -7 M PAF and 20 X 106 cells. An equivalent volume of 0.9% NaC1 containing bovine serum albumin (2.5 mg/ml) was included in control tubes. Incubations were started by adding 20 X 106 cells to each tube and were performed at 37~ Measurement of fatty acid uptake. Incubations were terminated by adding 5 ml ice-cold PBS containing fatty acid-free serum albumin {4 mg/ml} and 0.2 mM phloretin (Sigma} (stop solution} to each tube. The tubes were immediately centrifuged at 400 • g for three minutes at 4~ An aliquot of the supernatant solution was removed for counting radioactivity by liquid scintillation spectrometry. In zero time incubations, cell suspension was added after stop solution had been delivered to the tube. Fatty acid uptake is expressed as a percent of total radioactivity remaining in the incubation medium after correction for zero time incubation. Phospholipid extraction and analysis. Incubations were terminated by adding 5 ml methanol to each tube. Total lipids were extracted according to the method of Bligh and Dyer (14) and dissolved in chloroform] methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. Individual phospholipids were resolved by two-dimensional thin-layer chromatography (TLC) and analyzed as previously described (15). Since the basal levels of phosphatidic acid from as many as 20 X 106 neutrophils could not be accurately measured by phosphate assay, the radioactivity of individual phospholipids is expressed as dpm/6 X 106 cells.

Hydrolysis of phospholipids by phospholipase A~ Phosphatidic acid (PA}, PI and PC were each extracted from the thin-layer chromatography plate as described previously (15). For the identification of PA on the TLC plates, standard PA from egg yolk lecithin (Sigma) was applied together with neutrophil lipid extract to each TLC plate. Phospholipase Az hydrolysis of phospholipids was performed according to the method of Brockerhoff {16), with modification. Each phospholipid (0.5 to 1.5 t~g phosphorus) was dissolved in 1.0 ml diethyl ether and was incubated with 10 ~l (20 ~g protein} porcine pancreatic phospholipase Ae {Sigma) in the presence of 0.1 ml of 0.1 M MOPS (morpholinopropanesulfonic acid} buffer containing 1 mM CaC12, pH 7.2. Hydrolysis of phospholipids was carried out for 18 hr at room temperature. It was terminated by adding 0.5 ml of 2% acetic acid in ethanol. The precipitated salt was removed by brief centrifugation and was washed once more with the same solution. The combined ethanol solution was evaporated to dryness, and the lipid residue was redissolved in chloroform-methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene. Fatty acids and lysophospholipids were separated by thinlayer chromatography in a solvent system consisting of chloroform/methanol/28% ammonia (25:10:2, v/v/v). Radioactivity in the fatty acid and lysophospholipid bands was measured by scraping the gel into vials and counting by liquid scintillation spectrometry. Mild alkali hydrolysis of [14C]palmitic acid-labeled

PC. Each [~4C]palmitic acid-labeled PC fraction (1.5 ~g phosphorus) from resting and PAF-stimulated neutrophils was dissolved in 0.5 ml of chloroform and mixed with 0.5 ml of 0.2 N NaOH in methanol. The hydrolysis was conducted at room temperature for 1 hr as described by Rider et al. {17}. The samples were then neutralized with 1 N hydrochloric acid and extracted with the solvent system of Bligh and Dyer (15}. The resulting fatty acids and ether-linked lysoPC fractions were resolved by TLC, and the radioactivity in each fraction was measured by liquid scintillation spectrometry. The distribution of labeled fatty acid in the diacyl and alkylacyl PC was analyzed as previously described 115). RESULTS

Effect of P A F on fatty acid uptake. Previous studies {12) demonstrated a maximum stimulatory effect by PAF at 10 -7 M on phospholipid acylation by arachidonic acid, hence in the present study, 10-v M PAF was used in all experiments. Table 1 demonstrates that the percent of total radiolabeled fatty acid remaining in the incubation medium was decreased in the presence of PAF, indicating an increased uptake of fatty acids by neutrophils. All three fatty acids tested became rapidly cell associated with a corresponding loss of label from the incubation medium. A wide variation was found among different donors in the rate of loss of label from the incubation medium for all three fatty acids. This was also reported for arachidonic acid uptake by resting rabbit neutrophils {18}. However, PAF consistently increased the loss of label from the incubation medium at all time intervals (1-10 min).

Time course of P A F effect on the incorporation of [1-14C]linoleic acid and [1-14Clpalmitic acid into phospholipids. Figure 1 demonstrates the time course of the effect of PAF on the incorporation of linoleic acid into phospholipids. PAF inhibited the incorporation of [1-~4C]linoleic acid into PA, but it enhanced the incorporation of this fatty acid into PI and PC. In two separate experiments, the average radioactivities of PI and PC were increased to 1980 and 600% of control after 1 and 10 min incubations, respectively. The magnitude of stimulation (percentage of control} on the formation of labeled PI and PC varied with incubation time. The labeling of phosphatidylethanolamine (PE} and phosphatidylserine (PS) by [1-a4C]linoleic acid was not influenced by the presence of PAF even after a 10-min incubation. Analysis of the phosphorus content of the major phospholipid classes including PC, PE, PI, PS and sphingomyelin revealed no measurable changes during neutrophil-PAF interaction. Figure 2 shows the time course of the effect of P A F on the incorporation of [1-14C]palmitic acid into phospholipids. PAF promoted the incorporation of [1-14C]palmitic acid most significantly into PC. In two separate experiments, the average radioactivity of PC was increased to 825 and 134% of control after 1 and 10 rain incubations, respectively. An increased incorporation of palmitic acid into PI in the presence of PAF was measurable after a 5-min incubation. The stimulation by PAF of palmitic acid UPIDS, Vol. 24, No. 9 (1989)

814

J.-S. TOU TABLE 1 Time Course of the Effect of PAF on the Uptake of Fatty Acids by Human Neutrophils

Percent of total radiolabeled fatty acid remaining in the incubation mediuma Incubation Arachidonate Linoleate Palmitate minutes Control PAF Control PAF Control PAF 1 91• 85• 89• 83• 95• 86• 2 78• 65• 75• 64• 85• 72_4.4 5 43• 35• 38• 28• 67+-4.7 48• 10 24• 19• 15• 1.8 12• 29• 20• aNeutrophils (20 X 10 6) w e r e incubated at the indicated period of time with 0.1 ~Ci [1J4C]arachidonic acid (0.97 ~M), or 0.125 ~Ci [1-14kC]linoleic acid {1.14 ~M), or 0.1 gCi palmitic acid (0.95 ~M) in the absence or presence of 10-7 M PAF. Incubation conditions and measurement of fatty acid uptake were described in Materials and Methods. The data are means + SD from four separate experiments.

o ~ o Control :

: 10-7M

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~

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of I n c u b a t i o n

FIG. I. Time course of the effect of PAF on the incorporation of [l-14C]linoleic acid (18:2 In-6]) into phospholipids by human neutrophils. Neutrophils (20 X 106) were incubated at the indicated period of time (I-I0 min) with 0.125 ~Ci [l-14C]linoleic acid in the absence ( O ) or presence ( e ) of 10 -7 M PAF. Each point represents the average value of duplicate incubations from two separate neutrophil preparations. PA, phosphatidie acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.

i n c o r p o r a t i o n i n t o P A w a s less p r o n o u n c e d t h a n t h a t into PC. A f t e r a 1-min i n c u b a t i o n the radioa c t i v i t y of P A was increased to 153% of control, b u t it returned to basal level after a 10-min incubation.

Positional distribution of radiolabeled linoleic acid and palmitic acid in phospholipids. Extracellular linoleic acid and palmitic acid have been found to be incorporated b y neutrophils into b o t h sn-1 and sn-2 positions of PC {19,20). If the increased incorporation of these f a t t y acids into P I and PC in the presence of P A F is caused only b y increased p h o s p h o l i p a s e A2 activity, t h e n the p e r c e n t a g e of labeled f a t t y acids in t h e sn-2 p o s i t i o n of p h o s p h o l i p i d s f r o m P A F s t i m u l a t e d cells would be g r e a t e r t h a n t h a t f r o m control cells. The r e s u l t s show t h a t the positional d i s t r i b u t i o n of neither labeled f a t t y acid in PA, P I a n d PC w a s a f f e c t e d b y t h e p r e s e n c e of P A F . After t r e a t m e n t of each phospholipid with pancreatic p h o s p h o l i p a s e A2, 79, 92 and 74% of radiolabeled linoleic acid w a s r e l e a s e d f r o m PA, P I a n d PC, r e s p e c t i v e l y . 2-Min a n d 10-min i n c u b a t i o n s exLIPIDS,Vol. 24, No. 9 (1989)

h i b i t e d i d e n t i c a l p o s i t i o n a l d i s t r i b u t i o n of radiolabeled linoleic acid for each phospholipid. The positional distribution of radiolabeled palmitic acid in phospholipids was found to v a r y with incubation time. After a 2-min incubation, 72, 85 and 78% of radiolabeled palmitic acid in PA, P I and PC, respectively, were found in the sn-2 position, whereas after a 10-min incubation the radiolabeled palmitic acid in the sn-2 position became 60, 67 and 53% in PA, P I and PC, respectively. P h o s p h o l i p i d s isolated f r o m P A F s t i m u l a t e d n e u t r o p h i l s d e m o n s t r a t e d a similar positional distribution for b o t h f a t t y acids.

Effect of P A F on the distribution of labeled fatty acids in diacyl and alkylacyl PC. Neutrophil PC has a high content of alkylacyl species (21,22), and the sn-2 position of endogenous alkylacyl PC contains linoleic acid and palmitic acid besides arachidonic acid {21). Thus it is i m p o r t a n t to examine whether P A F alters the distribution of labeled f a t t y acids in the diacyl and alkylacyl PC. The results showed t h a t P A F stimulated the incorporation of extracellular f a t t y acids into b o t h diacyl and alkylacyl PC. While more radioactivity was

815

PAF AND PHOSPHOLIPID ACYLATION TABLE 2 Control

Effect of PAF on the Distribution of [1-14C]Arachidonic Acid in Diacyl and Alkylacyl Phosphatidylchollne (PC)

~. 1 0 - 7 M PAF

Radioactivity (dpm X 103)a -Pl m

Incubation Diacyl PC Alkylacyl PC minutes Control PAF Control PAF 2 0.81 • 0.2 2.12 • 0.3 0.11 -t= 0.02 0.55 + 0.1 10 2.52 • 0.4 3.0 • 0.3 0.34 :|: 0.1 0.75 • 0.1 aNeutrophils (20 X 106)were incubated with 0.1 ~Ci [1-14C]arachidonic acid {0.97 gM} for 2 and 10 min in the absence and presence of 10 -7 M PAF. Each PC fraction isolated from 60 X 106 cells was resolved into diacyl and alkylacyl species as described in Materials and Methods. The radioactivity in each species was derived from 1.5 ~g phosphorus of PC, and represents the mean • SD from three experiments.

5

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Effect of P A F on the Distribution of [1-14C]Linoleic Acid in Diacyl and Alkylacyl Phosphatidylcholine (PC)

Radioactivity (dpm X 103)a 1

0

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0

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Minutes of Incubation

FIG. 2. Time course of the effect of PAF on the incorporation of [1-14C]palmitic acid (16:0) into phospholipids by human neutrophils. Neutrophils {20 X 106) were incubated at the indicated period of time (1-10 min) with 0.1 [1-]4C]plamitic acid in the absence ( O ) or presence ($) of 10 -7 M PAF. Each incubation represents the average value of duplicate incubations from two separate neutrophil preparations. PA, phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol.

Incubation Diacyl PC Alkylacyl PC minutes Control PAF Control PAF 2 0.78• 3.0• 0.02• 1.8• 10 5.2 • 5.4• 0.19• 2.0• aNeutrophils(20 X 106)wereincubatedwith0.125gCi[1A4C]arachidonic acid {1.14 gM) for 2 and 10 min in the absence and presence of 10 -7 M PAF. Each PC fraction isolated from 60 X 106 cells was resolved into diacyl and alkylacyl species as described in Materials and Methods. The radioactivity in each species was derived from 1.5 gg phosphorus of PC, and represents the mean • SD from three experiments. TABLE 4 Effect of PAF on the Distribution of [1A4C]Palmitic Acid in

Diaeyi and Alkylacyl Phosphatidyleholine (PC) Radioactivity (dpm X 103)a r e c o v e r e d in t h e d i a c y l species, t h e m a g n i t u d e of inc r e a s e of r a d i o a c t i v i t y in t h e a l k y l a c y l s p e c i e s w a s m o r e p r o n o u n c e d t h a n t h a t in t h e d i a c y l species. E a r l i e r s t u d i e s (12) o n l y d e m o n s t r a t e d a n i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d a r a c h i d o n i c a c i d i n t o t h e diacyl s p e c i e s of P C a f t e r a 1-min i n c u b a t i o n of n e u t r o p h i l s w i t h P A F . T h e p r e s e n t s t u d y {Table 2) d e m o n s t r a t e d t h a t a f t e r a l o n g e r p e r i o d of i n c u b a t i o n , P A F s t i m u l a t e d t h e i n c o r p o r a t i o n of a r a c h i d o n i c a c i d i n t o b o t h d i a c y l a n d a l k y l a c y l PC. A n a l y s i s of t h e f o r m a t i o n of a r a c h i d o n i c a c i d - l a b e l e d d i a c y l a n d a l k y l a c y l s p e c i e s a t 2 a n d 10 rain i n d i c a t e d t h a t t h e i n c r e m e n t of a r a c h i d o n i c a c i d l a b e l e d d i a c y l s p e c i e s i n d u c e d b y P A F w a s m o r e e v i d e n t a f t e r t w o m i n t h a n a f t e r 10 m i n of i n c u b a t i o n . T h e f o r m a t i o n of l a b e l e d a l k y l a c y l species w a s n o t f u r t h e r i n c r e a s e d a t 10 m i n in t h e p r e s e n c e of P A F . A s s h o w n in T a b l e 3, in r e s t i n g n e u t r o p h i l s e x o g e n o u s linoleic a c i d w a s m a i n l y i n c o r p o r a t e d i n t o d i a c y l P C a n d o n l y a b o u t 3% of t h e t o t a l r a d i o a c t i v i t y in P C w a s r e c o v e r e d in t h e a l k y l a c y l s p e c i e s a f t e r a 10 m i n incubation. In PAF-stimulated neutrophils there were about 39% a n d 23% of t h e t o t a l r a d i o a c t i v i t y in PC b e i n g r e c o v e r e d in t h e a l k y l a c y l s p e c i e s a f t e r 2 a n d 10 m i n i n c u b a t i o n s , r e s p e c t i v e l y . T h e i n c r e m e n t in t h e forma-

Incubation Diacyl PC Alkylacyl PC minutes Control PAF Control PAF 2 1.0• 4.2• 0.28• 2.5+0.4 10 9 . 2 • 1.2 11 • 1.5 1.28• 3.4• aNeutrophils (20 X 106)were incubated with 0.1 ~Ci [1-14C]arachidonic acid (0.97 ~M) for 2 and 10 min in the absence and presence of 10 -7 M PAF. Each PC fraction isolated from 60 • 106 cells was resolved into diacyl and alkylacyl species as described in Materials and Methods. The radioactivity in each species was derived from 1.5 ~g phosphorus of PC, and represents the mean • SD from three experiments. t i o n of linoleic a c i d - l a b e l e d a l k y l a c y l P C i n d u c e d b y P A F a p p e a r e d t o h a v e r e a c h e d a m a x i m u m b e f o r e 10 min, a s a l k y l a c y l P C e x h i b i t e d s i m i l a r r a d i o a c t i v i t y a f t e r i n c u b a t i o n of cells w i t h P A F for 2 a n d 10 min. I n t h e p r e s e n c e of P A F , t h e r a d i o a c t i v i t y of linoleic a c i d - l a b e l e d d i a c y l P C b e c a m e 400% of c o n t r o l a t t w o m i n u t e s , b u t i t r e m a i n e d t h e s a m e a s t h a t in c o n t r o l cells a t 10 min. T h e e f f e c t o f P A F o n t h e d i s t r i b u t i o n of l a b e l e d p a l m i t i c a c i d in t h e d i a c y l a n d a l k y l a c y l P C is s h o w n in T a b l e 4. I n r e s t i n g n e u t r o p h i l s 20 a n d 12% of t o t a l P C r a d i o a c t i v i t y w e r e r e c o v e r e d in t h e a l k y l a c y l speLIPtDS,Vol, 24, No, 9 (1989)

816

J.-S. TOU cies after 2 and 10 min incubation, respectively. In PAF-stimulated cells, a 2-min incubation with [1-14C]palmitic acid resulted in an increase of labeled alkylacyl PC to 772% of control and of labeled diacyl species to 352% of control. After 10 min incubation of cells with PAF and [1-14C]palmitic acid, the radioactivity of alkylacyl PC was increased to 264% of control, and that of diacyl species became 122% of control. Mild alkaline hydrolysis of palmitic acid-labeled PC. This experiment was carried out to examine if the radioactivity in palmitic acid-labeled alkylacyl PC was solely derived from the acyl moiety and not from the alkyl moiety. After deacylation of PC with methanolic NaOH, the resulting fatty acids and 1-alkyl-sn-glycero3-phosphocholine (lysoPAF) were separated by TLC. It was found that the radioactivity was completely recovered in the f a t t y acid fraction and no radioactivity was detected in ether-linked lysophosphatidylcholine.

(d) acyltransferase activity. If the increased phospholipid acylation induced by PAF is solely secondary to increased phospholipase A 2 activity, the percentage of radioactivity in the sn-2 position of PI and PC from PAF-stimulated cells would be greater than that from control cells. However, PAF was not found to alter the positional distribution of labeled fatty acids in PI and PC after incubation of neutrophils for either 2 or 10 min. Since there were no detectable changes in the mass of PI and PC during PAF-neutrophil interaction, both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation by exogenous fatty acids. Indeed, an activation of phospholipase A 2 by exogenous PAF was demonstrated in cytocholasin Bprimed rabbit neutrophils {26}. The incorporation of exogenous palmitic acid into the sn-2 position of PC was also reported in human neutrophils {20}. The present study demonstrated a variation of the positional distribution of palmitic acid in phospholipids with incubation time. More labeled palmitic acid being recovDISCUSSION ered in the sn-2-position after a 2-min incubation may The present study extended the earlier studies {12}, indicate a higher basal level of 1-acyl-2-1ysophosand demonstrated that PAF stimulates the uptake of pholipids than that of 1-1yso-2-acyl-phospholipids in fatty acids from extracellular medium. It also demon- neutrophils, and more labeled palmitic acid being recovstrated that PAF increases the incorporation of lino- ered in the sn-1 position after a longer period of incubaleic acid and palmitic acid into PI and PC. In addition, tion may indicate a shift to a de novo pathway. the present study illustrated that PAF enhances the Although neutrophil alkylacyl PC has a high conincorporation of extracellular fatty acids into both dia- tent of linoleic and palmitic acid {21}, rabbit neutrocyl and alkylacyl PC. phils incorporated these two fatty acids only into the In the absence of competing labeled fatty acids, diacyl species (20). The present study also demonstrated human neutrophils did not appear to exhibit a prefer- that resting human neutrophils incorporated linoleic ential uptake of arachidonic acid. Linoleic acid and acid mainly into the diacyl species of PC. Although the palmitic acid also rapidly became cell associated. The present study demonstrated that resting human neumechanism by which fatty acid enters into neutrophils trophils incorporated a significant amount of exogenous has not yet been defined. It may be a transport process palmitic acid into alkylacyl PC, the incorporation of or a simple diffusion. Due to the rapid incorporation this f a t t y acid into alkylacyl PC is expected to be of fatty acids into phospholipids, it is difficult to sepa- attenuated by the presence of arachidonic acid in the rate the uptake from activation and esterification of incubation medium. The increased incorporation of fatty fatty acids under the experimental conditions. How- acids into alkylacyl PC in PAF-stimulated neutrophils ever, the finding that PAF promotes the loss of labeled could be due to increased lysoPAF. LysoPAF can be fatty acids from the incubation medium suggests an derived from endogenous alkylacyl PC after an activaincreased fatty acid uptake during PAF-neutrophil in- tion of phospholipase A 2 during PAF-neutrophil interteraction. It is not known whether the increased fatty action; it can also be derived from PAF after deacetylaacid uptake is specific for PAF among the agonists for tion (27}. neutrophil activation. There is evidence for increased An increased incorporation of exogenous fatty acPAF formation in neutrophils in response to formyl- ids into PI and PC by neutrophils in response to PAF methionyl-leucyl-phenylalanine(1,7}, leukotriene B 4 (23}, may serve to replenish these phospholipids following ionophore A23187 {24}, and phorbol esters {25}. Inter- deacylation. This may also divert exogenous arachidonic pretation of fatty acid uptake by neutrophils in the acid from the 5-1ipoxygenase pathway, thereby attenupresence of any one of these agonists would be compli- ating the formation of leukotriene B4 (LTB4) and 5cated by increased PAF formation under these condi- hydroxyeicosatetraenoic acid (5-HETE), both of which tions. Parallel studies on fatty acid uptake and PAF are mediators of inflammation (28,29}. Furthermore, formation would be required to ascertain the specific increased acylation of lysoPAF induced by PAF would effects of these agonists on fatty acid uptake. allow a rapid termination of further synthesis of PAF, The magnitude of stimulation by PAF on the for- as it has been shown that PAF stimulates its own mation of labeled PI and PC appears to reflect the synthesis in human neutrophils {23,30}. It remains to specificity of the acyltransferases catalyzing the acyl- be tested if exogenous palmitic acid and linoleic acid ation of the respective lysophospholipids. It was greater are more effective than arachidonic acid in attenuating in PI than in PC for the incorporation of linoleic acid PAF synthesis by PAF-stimulated neutrophils, since and vice versa for the incorporation of palmitic acid. activated neutrophils may metabolize part of the exIncreased phospholipid acylation could be brought about ogenou~ arachidonic acid to LTB 4 and 5-HETE, both by increased: (a) fatty acid uptake; (b} phospholipase of which have been shown to increase PAF synthesis A2 activity; (c) fatty acyl-CoA synthetase activity; and (23}. LIPIDS,Vol. 24, No. 9 (1989)

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PAF AND PHOSPHOLIPID ACYLATION

ACKNOWLEDGEMENT This work was supported by research grants from the American Heart Association-Louisiana, Inc., and from the Cancer Association of Greater New Orleans, Inc.

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