Aug 20, 1990 - 6928-6935, 1991. Printed in U. S. A. Metabolism ...... F. (1985) J. Bid. Chern. 24. Kucera, G. L., Sisson, P. J., Thomas, M. J., and Waite, M. (1988).
THEJOURNALOF BIOLOGICAL CHEMISTRY 1991 by The American Society for Biochemistry andMolecular Biology, Inc.
Val. 266, No. 11, Issue of April 15. pp. 6928-6935, 1991 Printed in U.S. A .
1 4
Metabolism of l-Acyl-2-acetyl-sn-glycero-3-phosphocholine in the Human Neutrophil* (Received for publication, August 20, 1990)
Massimo Triggiani, DeborahM. D’Souza, and Floyd H. Chilton$ From The Johns Honkins Asthmaand Allern Center, The Johns Hopkins University School of Medicine, -” Baltimore, Maryland 21224
Thebiosynthesis of l-acyl-2-acetyl-sn-glycero-3-containinganacetate moiety at the sn-2 position of the phosphocholine(1-acyl-2-acetyl-GPC)togetherwith glycerol backbone (2-acetylatedphospholipids).l-Alkyl-2that of 1-alkyl-2-acetyl-GPC (platelet-activating fac- acetyl-sn-glycero-3-phosphocholine (1-alkyl-2-acetyl-GPC)’ tor) has been demonstrated in a variety of inflamma- was first identified andtermedplatelet-activatingfactor tory cells and tissues. It hasbeen hypothesizedthat the (PAF) because of its ability to induce platelet aggregation (for relative proportion of these phospholipids produced review, see Refs. 1 and 2). More recently, the biosynthesis of upon cell activation may be influenced by their rates other2-acetylated phospholipids has been describedina of catabolism. We studied the catabolism of l-acyl-2- variety of cells and tissues. These phospholipids include 1acetyl-GPC in resting and activated human neutrophils acyl-2-acetyl-GPC (3-6), l-alk-l’-enyl-2-acetyl-GPC ( 7 ) ,and and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC 1- alk- 1’-enyl - 2-acetyl-sn-glycero- 3-phosphoethanolamine and 1-acyl-2-acetyl-GPC; however, the rate of catab- (8).Each cell or tissue appear to produce a unique subset of olism of 1-acyl-2-acetyl-GPC is approximately 2-fold these phospholipids. For example, the human neutrophil has higher than thatof 1-alkyl-2-acetyl-GPC. In addition, been shown to produce large quantities of 1-alkyl-2-acetyll-alk-l’-enyl-2-acetyl-sn-glycero-3-phosphoethamost of 1-acyl-2-acetyl-GPC is catabolized through a GPCand of 1-acyl-2-acetyl-GPCwhen pathway different from thatof 1-alkyl-2-acetyl-GPC. nolamine and smaller quantities The main step in the catabolism of 1-acyl-2-acetyl- stimulated by a calcium ionophore (A23187) (3, 4). In con(9) and murine GPC is the removal of the long chainat the sn-1posi- trast, we have recently found that human mast cells produce larger quantities of 1-acyl-2-acetyl-GPC tion; the long chain residue is subsequently incorporelative to other %acetylated phospholipids upon immunorated either into triglycerides or phosphatidylchointo be line. The 1-lyso-2-acetyl-GPC formed in this reaction logic activation. Similarly, 1-acyl-2-acetyl-GPC appears to is thenfurtherdegradedto glycerophosphocholine, the major %acetylated phospholipid synthesized by A23187choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is stimulated murine macrophages (10). Although the biological also catabolized, toa lesser extent, through deacetyla- activity of PAF has been extensively studied, the role of the tion at the sn-2 position and reacylation with a long other 2-acetylated phospholipids as mediators of cellular rechain fattyacid. Stimulationof neutrophils by A23187 sponses has yet to be fully understood. 1-Acyl-2-acetyl-GPC results in a higher rate of catabolism of 1-acyl-2-ace- has beenshown to be approximately 200-1,OOO-fold less active tyl-GPC by increasing both the removal of the long than PAF in inducing the degranulationof human neutrophils chain at the sn-1 position and the deacetylation-reand asa hypotensive agent in the rat(11, 12). In addition, we acylation at the sn-2position. In a broken cell prepa- found that 1-acyl-2-acetyl-GPC may act asa specific inhibitor ration, the cytosolic fraction of the neutrophil was of PAF-induced activation of the human neutrophil.’ This shown to contain an enzyme activity whichcleaved the observation suggests that the potential proinflammatory role sn-1 position of 1-acyl-2-acetyl-GPCandl-acyl-2of a cell may be dependent not only on the amount but also lyso-GPC but not of l,%-diacyl-GPC. Taken together, these data demonstrate that the human neutrophil is on the typeof 2-acetylated phospholipids that areproduced. The nature and quantities of the 2-acetylated phospholipids able to catabolize 1-acyl-2-acetyl-GPC in a manner that can be measured in a cell depends in turn on a number both quantitatively and qualitatively different from of factors which include the activity of the enzymes and the that of platelet-activating factor. The differential catabolism may regulate the relative proportion of these availability of precursors utilized in the biosynthesis of these phospholipids. An equally important factor is the catabolic two bioactive phospholipids in the neutrophil. rate by which the 2-acetylated phospholipids can be converted to metabolites. For example, Sturk and colleagues (14) have recently determined that, in the presence of anonspecific It has become apparent in the last few years that cells inhibitor of the catabolism, phenylmethylsulfonylfluoride, involved in inflammatoryprocesses such as neutrophils, mac- the human neutrophils produce larger quantities of l-acyl-2rophages, and mast cells produce a variety of phospholipids acetyl-GPC relative to that of 1-alkyl-2-acetyl-GPC. In addition, they demonstrated that human platelets produce almost * This work was supported inpart by National Institutesof Health
Grants AI 24985 and AI 26771. This is publication No. 045 from the Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224. T h e costs of publication of this article were defrayed in part by the be hereby payment of page charges. Thisarticlemusttherefore marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.
The abbreviations used are: l-alkyl-2-acetyl-GPC, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine;GPC, glycero-3-phosphocholine; HBSS, Hank’s balanced salt solution; HSA, essentially fatty acid free PC, phosphatidylcholine;TLC,thinlayer humanserumalbumin; chromatography; PAF, platelet-activating factor. ’ M. Triggiani, D. W. Goldman, F. H. Chilton, manuscript submitted.
6928
Metabolism of 1 -Acyl-2-acetyl-GPC
6929
exclusively 1-acyl-2-acetyl-GPC when stimulated with throm-cytosolic and membrane compounds by ultracentrifugation (100,000 g, 45 min, 4 "C). The supernatant (cytosolic fraction) was removed, bin in the presence of phenylmethylsulfonyl fluoride. These x and the pellet (membranefraction) was resuspended in 10 ml of data suggest that the profile of %acetylated phospholipids HBSS. Two-hundred pl of the cytosol and membrane preparation detected in inflammatory cells is influenced in large part by were added to 800 pl of HBSS containing 1.2 mM Ca2+ and2 nmol of l-[14C]acyl-2-acetyl-GPC, l-[14C]acyl-2-lyso-GPC,or l-['4C]2-diacylthe catabolism of these molecules. The catabolism of PAF hasbeen studied in detail in several GPC complexed t o HSA (final concentration,1 mg/ml) and incubated cell types. Removalof the acetategroup by an acetyl hydrolase a t 37 "C for 30 min. The reactions were terminated by the addition of methanol/chloroform (21,v/v), and the lipids were extracted from has been found to be the first step in the catabolism of PAF the reaction mixtureby the method of Bligh and Dyer (22). within inflammatory cells (15-18). The resulting l-alkyl-2Analysis of Products Derived from 1-Alkyl-2-acetyl-GPC and 1lyso-GPC is then rapidly reacylated at the sn-2 position with Acyl-2-acetyl-GPC Catabolism-The labeled compounds derived from 1-0-[3H]alkyl-2-acetyl-GPC and l-[14C]acyl-2-acetyl-GPCwere sepunsaturatedfatty acids. Arachidonate is thepredominant fatty acid chain incorporated into 1-alkyl-2-lyso-GPC by sev- arated by TLC on layers of Silica Gel G developed in chloroform/ eral cells (15-18). This pathway provides an effective mech- methanol/acetic acid/water (50:25:84, v/v). Labeled neutral lipids derived fromcatabolism of l-['4C]acyl-2-acetyl-GPC were further anism by which PAF levels are regulated by the cell. In separated by TLC on Silica Gel G plates developed in hexane/ethyl contrast to PAF, little is known about the metabolism of other ether/formic acid (90:60:6, v/v). 2-acetylated phospholipids in inflammatory cells. However, In the experiments inwhich the cells were incubated with l-acylwas counted in both the aqueous the aforementioned studies using phenylmethylsulfonyl fluo- 2-acetyl-[I4C]GPC, the radioactivity ride suggest that %acetylated phospholipids such as PAF andand the organic phase after lipid extraction. In addition, the radioactive products in the aqueous phasewere concentrated and further 1-acyl-2-acetyl-GPC may be catabolizedby different pathanalyzed by TLC on Silica Gel G plates developed in ethanol/water/ ways. In the present study we have defined the biochemical ammonium hydroxide (4870:7, v/v). This system separates waterreactions involved in the catabolism of 1-acyl-2-acetyl-GPC soluble choline metabolites including choline ( R p , 0.08), phosphochoin resting and stimulated human neutrophils. line (RE.,0.34), glycerophosphocholine (Rh0.57), and 1-lyso-2-acetylglycerophosphocholine (RF,0.67). The radioactivelipids were detectedontheTLCplates by a System 200 Imaging Scanner (Washington, D. C.). PhosphoMaterials-l=O-[~'H]Alkyl-2-acetyl-GPC (60.0 Ci/mmol; contain- Bioscan lipid and neutral lipid standards were identified by I, vapors and the ing >96%1-hexadecyl) and 1,2-dipalmitoyl-["C]GPC (I4C-methyl, corresponding areas were scraped into vials for liquid scintillation 153 mCi/mmol) were obtained from Du Pont-New England Nuclear. counting. l-['4C]Palmitoyl-2-lyso-GPC (55 mCi/mmol) was obtainedfrom Amersham Corp. PAF, lyso PAF, essentially fatty acid free human RESULTS serum albumin (HSA)were obtained fromSigma. 1-Palmitoyl-2-lysoGPC was obtained from AvantiPolar Lipids (Birmingham,AL). Catabolism of J-[3HIAlkyl-2-acetyl-GPC and 1-[14ClAcylIonophore A23187 was purchased from Calbiochem. Acetic anhydride neutrophils stimulated with A23187 and pyridine were obtained from Aldrich. All solvents (high perform- 2-acetyl-GPC-Human (2 p ~ in) the presenceof ["]acetate incorporated thelabeled ance liquid chromatography grade) were obtained from Fisher. Preparation of Phospholipids-1-Palmitoyl-2-acetyl-GPC(l-acyl- acetate into %acetylatedphospholipids (3,463 k 955 dpm/106 2-acetyl-GPC) and l-[1'C]palmitoyl-2-acetyl-GPC were prepared by cells; mean k S.E.; n = 3). 1-Acyl-2-acetyl-GPC accounted acetylation of 1-palmitoyl-2-lyso-GPC and of l-[14C]palmitoyl-2-lys~for only 9.5 k 1.5% of these labeled phospholipids, the reGPC, respectively, in the presence of pyridine and acetic anhydride maining being constituted by PAF. Thisobservation led us to (19). I-Palmitoyl-2-a~etyl-['~C]GPC was prepared by phospholipase A? hydrolysisof 1,2-dipalmitoyl-["C]GPC and subsequent acetylation investigate if a differential catabolismcould be a factor deterof l-palmitoyl-2-lyso-['4C]GPC (14). The acetylated compounds were mining the ratio of the two phospholipids in the neutrophil. Initial experimentswere performed to compare the catabolism purified by TLC onSilicaGelG plates developed in chloroform/ of exogenous 1-alkyl-2-acetyl-GPC (PAF)with that of l-acylmethanol/aceticacid/water (50:25:8:4, v/v). l-Lyso-2-acetyl-[''C] GPC was prepared by lipaseAI(Rhizopus arrhyzus,Boehringer 2-acetyl-GPC in the resting neutrophils. Fig. lA shows that Mannheim, West Germany) hydrolysis of l-palmitoyl-2-a~etyl-['~C]human neutrophils rapidly catabolize both 1-alkyl-2-acetylGPC (20). [14C]Phosphocholine and ['4C]glycerophosphocholine were GPC and 1-acyl-2-acetyl-GPC;moreover, the rate of cataboprepared by phospholipase C hydrolysis (Type XIII, from Bacillus pmol/min/107 cereus, Sigma) (19) andmild alkaline hydrolysis (19), respectively, of lism of 1-acyl-2-acetyl-GPC(initialrate,16 cells) was approximately 2-fold higher than that of l-alkyl-21,2-dipalmitoyl-["C]GPC. Incubation Conditions and Extraction of Lipids-Neutrophils were acetyl-GPC (initial rate, 8.3 pmol/min/107 cells). In another isolated by dextran sedimentation followed by Ficoll-Paque density group of experiments, the rateof catabolism of both l-alkylgradient centrifugation as previously described (21). Isolated neutro- 2-acetyl-GPC and 1-acyl-2-acetyl-GPC was determined when phils were resuspended in HBSS withcalcium (1.2 mM) prior to their various amounts of the two phospholipids were added to the use in each experiment. The neutrophils (10' cells/tube) were then Fig. 1B confirm that the rate incubated a t 37 "C in a volume of 1 ml for various periods of time in reaction mixture. The results in the presence of l-[:'H]alkyl-2-acetyl-GPC, l-['4C]acyl-2-acetyl-GPC, of catabolism of 1-acyl-2-acetyl-GPCwas higher than that of 1-alkyl-2-acetyl-GPC at all concentrations tested. They also or l-a~yl-2-acetyl-['~C]GPC (final concentration, 0.5 nmol/tube unless otherwise indicated) complexed to HSA (final concentration,0.05 show that the rate of catabolism of 1-acyl-2-acetyl-GPC inmg/ml).Sincethe radiospecific activity of I-O-[:'H]alkyl-2-acetyl- creased as a function of the amountof the phospholipid added GPC was 1,000-foldhigher than that of l-[14C]acyl-2-acetyl-GPC, to the cells. In contrast, the capacity of the neutrophils to unlabeled 1-0-alkyl-2-acetyl-GPC was addedto achieve the final catabolize 1-alkyl-2-acetyl-GPC increased to a maximal activconcentration of PAF.Intheexperimentswithstimulated cells, ity of approximately 10 pmol/min/lO' cells andremained A23187 ( 2 p ~ was ) added to thetubessimultaneouslywiththe concentrations of thesubstrate. phospholipids. Incubations were terminated by the addition of meth- constant withincreasing anol/chloroform (2:1, v/v) to the reaction mixture, and the lipids These datarevealed that the neutrophil had a greater capacity were extracted by a modified method of Bligh and Dyer (22) in which to catabolize 1-acyl-2-acetyl-GPC than 1-alkyl-2-acetyl-GPC. sufficient formic acid was added to lower the pHof the aqueouslayer In order to understand if there were differencesin the to 3. Recovered products included both cell-associated component metabolic pathway for 1-alkyl-2-acetyl-GPC andl-acyl-2and those present in the medium. Recovery of added radioactivity acetyl-GPC, the neutrophils were incubated with either 1after lipid extraction always exceeded 85%. ["H]alkyl-2-acetyl-GPCor 1-['4C]acyl-2-acetyl-GPCand the Subcellular fractions were obtained from neutrophils(4 X lo7cells) metabolites produced during catabolism were separated by suspended in 10 ml of HBSS and sonicated ( 3 X 30 s, Microson, Heating System, Farmingdale, NY). The sonicate was separated into TLC. Fig. 2 shows theseparationandthedistribution of EXPERIMENTALPROCEDURES
Metabolism of 1 -Acyl-2-acetyl-GPC
6930
A. o 1-alkyl-2-acetyl-CPC
1-acyl-2-acetyl-CPC
U
SO"
L.
040 81::
A.
72" 0 0
20
1 60
40
63
--
54"
Time (min)
45"
36" 27"
18"
0
1
2
3
4
5
6
360 T
Substrate (nanomoles) FIG. 1. A , time course of the catabolism of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC in the human neutrophil. Neutrophils (lo7 cells/tube) were incubated at 37 "C in 1 ml of HBSS containing 0.5 nmol of l-[:'H]alkyl-2-acetyl-GPC or l-['4C]acyl-2-acetyl-GPC complexed to HSA (final concentration 0.05 mg/ml). At different time points the reactions were terminated by the addition of methanol/ chloroform (2:1, v/v). The lipids were extracted and separated by TLC on layers of Silica Gel G developed in chloroform/methanol/ acetic acid/water (50:25:8:4, v/v). The percentage of recovered radioactivity remaining as 1-alkyl-2-acetyl-GPC or 1-acyl-2-acetyl-GPC was determined by zonal scraping and liquid scintillation counting. These data are presented as the mean & S.E. of four experiments. B, rates of catabolism of 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC a t different concentrations of each substrate. Neutrophils (lo7cells/ tube) were incubated for 15 min as described in A withdifferent concentrations of l-[3H]alkyl-2-acetyl-GPCor l-["C]acyl-2-acetylGPC. The reactions were terminated and the lipids were extracted and analyzedby TLC. The radioactivity remaininginl-alkyl-2acetyl-GPC or 1-acyl-2-acetyl-GPC was measured by zonal scraping and liquid scintillation counting. These data are expressed as picomoles of substrate(1-alkyl-2-acetyl-GPC or 1-acyl-2-acetyl-GPC) catabolized in 15 min by lo7 neutrophils and are presented as the mean rt S.E. of three experiments.
radioactivity in the major metabolites produced during the catabolism of l-[3HH]alkyl-2-acetyl-GPC (Fig. 2 A ) and of 1[14C]acyl-2-acetyl-GPC (Fig. 2B). As previously reported (15), l-["H]alkyl-2-acetyl-GPCwas catabolizedprimarily to l-['H] alkyl-2-acyl-GPC with little formation of the intermediate compound, l-["H]alkyl-2-lyso-GPC. Less than 5% of radioactivity was found in the neutrallipid region. In contrast, the predominant metabolite of l-['4C]acyl-2-acetyl-GPC migrated by TLC with the neutral lipid fraction. In addition, there was the formation of 1,2-[14C]diacyl-GPCand of small quantities of l-['4C]acyl-2-lyso-GPC. The distributionof radioactivity in the metabolites derived from catabolism of 1['4C]acyl-2-acetyl-GPC as a function of time of incubation is shown in Fig. 3A. At all time points examined the neutral lipid compound(s) were the major metabolites derived from 1-acyl-2-acetyl-GPC. In addition, there was also a time-dependent accumulation of 1,2-[14C]diacyl-GPC.No catabolism of l-[14C]acyl-2-acetyl-GPCwas observed in the absence of cells (data not shown). Further examination of the neutral lipid(s) produceduponcatabolism of 1-['4C]acyl-2-acetylGPC in a less polar solvent system indicated that the radio-
0.0
4.0
8.0
Dirtonce
12.0
16.0
20.0
(cm)
FIG. 2. TLC separation of labeled compounds derived from the catabolism of l-[3H]-alkyl-2-acetyl-GPC( A ) and 1-[14C]acyl-8-acetyl-GPC ( B ) in the human neutrophil. Neutrophils
(lo7 cells/tube) were incubated for 15 min with 0.5 nmol of 1-[3H] alkyl-2-acetyl-GPC or l-['4C]acyl-2-acetyl-GPC. The lipids were extracted and analyzed by TLC on Silica Gel G plates developed in chloroform/methanol/acetic acid/water (50:25:84, v/v). The radioactivityontheplate wasanalyzedfor 10min by anautomated scanner. These data are representativeof four separate experiments. activity in the neutral lipid fraction migrated primarily with free fatty acids and triacylglycerols. Fig. 3B shows the formation of these labeled neutral lipids as a function of incubation time; there was accumulation of the radioactivity in free fatty acid within the first5 min. At later time points, the radioactivity in free fatty acid was reduced concomitantly with the appearance of labeled triglycerides.The radioactivity associated with monoglycerides, diglycerides or 1-acyl-2-acetyl-glycerols did not exceed 5% of total radioactivity at any time point. These results suggested that the catabolism of 1acyl-2-acetyl-GPC notonly occurreda t a faster rate than that of 1-alkyl-2-acetyl-GPC butalso by a different catabolic pathway. In particular, it appeared that the main step in the catabolism of 1-acyl-2-acetyl-GPC was the removal of the long chain at the sn-1 position and its subsequent incorporation into triglycerides. Catabolism of I-Acyl-2-a~etyl-~~C]-GPC-In order todefine the reaction sequence for the catabolism of 1-acyl-2-acetylGPC, another setof experiments was performed using l-acyl-
6931
Metabolism of 1-Acyl-2-acetyl-GPC
of neutrophils with l-a~yl-2-acetyl-['~C]GPCresulted in a time-dependent appearance of radioactivity in the water phase of the Bligh and Dyer extraction (22). After 60 min of 1JdiaCyl-CPC incubation, the water soluble component(s) account for more 0 l-seyl-2-lySo-CPC than 60% of the initial radioactivity. In addition to thewatersoluble metabolite(s), -16% of the initial radioactivity was found in 1,2-diacyl-[14C]GPCafter 60 min. These data confirm that most of 1-acyl-2-acetyl-GPC is catabolized through the removal of the long chain at the sn-1position. However, 0 20 40 60 1-acyl-2-acetyl-GPC is also catabolized, to a lesser extent, Time (min) through a deacetylation-reacylation pathway. Further analysis of the water-soluble metabolites derived from the catabolism of l-a~yl-2-acetyl-['~C]GPCwas perfree ratty ad& B. formed by TLC(Table 11). At the early time points the 0 triglycerides majority of the water-soluble radioactivity was found to mi40 1 dlglycerides monoglycerides grate with l-lyso-2-acetyl-GPC. A t the later time points the radioactivity was associated with GPC, choline, and phosphocholine. The transient formation of an unidentified product which migrated in this TLC system with an RF of 0.87 was also observed. These data suggest that, during the catabolism of l-acyl-2-acetyl-GPC, 1-lyso-2-acetyl-GPC is initially formed by the removal of the fatty acyl chain at the sn-1 0 20 40 60 position of the molecule. This metabolic intermediate is then 'lime (min) further metabolized to glycerophosphocholine and, eventuFIG. 3. A , time course of the formationof labeled metabolites from ally, to choline and/or phosphocholine. l-[14C]acyl-2-acetyl-GPC in the human neutrophil. Neutrophils (lo' Effect of Cell Activation on the Catabolism of 1-Acyl-2-acetylcells/tube) were incubated with0.5 nmol of l-["C]acyl-2-acetyl-GPC. GPC-The synthesis of 2-acetylated phospholipids in the At different timepoints, the reactions were terminated by the addition neutrophil occurs during cell activation. Therefore, it is critof methanol/chloroform (2:1, v/v). The lipids were extracted and ical to understand the catabolism of the 2-acetylated phosanalyzed by TLC on Silica Gel G plates developed in chloroform/ methanol/acetic acid/water (50:25:8:4, v/v). The formation of labeled pholipids in stimulated neutrophils. To better understandthe metabolites was determined by zonal scraping andliquid scintillation effect of neutrophil activation on the catabolism of l-acyl-2counting. These data are expressed as the percentage of the total acetyl-GPC, neutrophils were incubated with 1-[14C]acyl-2radioactivity recovered after lipid extraction and are presented as the acetyl-GPC in the presence of ionophore A23187 (2 PM). mean f S.E. of four experiments. B, time course of formation of labeled neutral lipids derived from the catabolism of 1-['4C]acyl-2- Neutrophils stimulated in this mannerincreased their capacacetyl-GPC in the human neutrophil. Neutrophils (10' cells/tube) ity to catabolize 1-acyl-2-acetyl-GPC as compared with unwere incubated with 0.5 nmol of l-['4C]acyl-2-acetyl-GPC. At differ- stimulated cells (unstimulated, 16 pmol/min/107 cells; stiment time points the reactions were terminated by the addition of methanol/chloroform (21, v/v). The neutral lipids were extracted ulated, 19.3 pmol/min/lO' cells). In thisgroup of experiments, we also compared the rates of catabolism of 1-alkyl-2-acetyland analyzed by TLC on Silica Gel G plates developed in hexane/ ethyl ether/formic acid (90:606, v/v). The formation of labeled meGPC (PAF) and 1-acyl-2-acetyl-GPC in neutrophils stimutabolites was determined by zonal scraping and liquid scintillation lated with A23187 (Table 111).At the lower doses of l-alkylcounting. These data are expressed as the percentage of the total 2-acetyl-GPC (0.1 and 0.5 nmol), the rate of catabolism of radioactivity recovered after lipid extraction and are presented as the PAF is reduced in A23187-stimulated cells as compared to mean f S.E. of three experiments. control cells; in contrast, at higher concentrations of l-alkyl2-acetyl-GPC labeled in the choline moiety of the molecule. 2-acetyl-GPC the rate of catabolism in stimulated cells is 1.5If the reaction proceeded by the removal of the long chain at 1.8-fold higher than in control cells. A possible explanation the sn-1 position of the molecule, this would give rise to 1- for this phenomenon is that, at thelowest concentrations of lys0-2-acetyl-['~C]GPCwhichwouldbe water-soluble. The exogenous (labeled) PAF, endogenously produced PAF might results of these experiments are shown in Table I. Incubation compete for the catabolic sites and, therefore, mask the in-
A.
o
1-acyl-2-acelyl-CPC neutrallipids
TABLE I Conversion of 1 -acyl-2-acetyl-["CIGPC to products Neutrophils (10' cells/tube) were incubated with 0.5 nmol of l-a~yl-2-acetyl-['~C]GPC. At different time points the reactions were stopped, and thelipids were extracted by the method of Bligh and Dyer (22). The radioactivity was measured in both the organic and the aqueous phase. The metabolites contained in the organic phase were then separated by TLC on Silica Gel G plates developed in chloroform/methanol/acetic acid/water (5025:8:4, v/ v). The labeled metabolites were determined by zonal scraping and liquid scintillation counting. These data are expressed as the percentageof the total radioactivityrecovered in both theorganic and the aqueous phase. These data are presented as the meanf S.E. of three exDeriments. % of total radioactivity Time 1-Acyl-2-acetyl-GPC 1-Acyl-2-lyso-GPC 1,2-Diacyl-GPC Water-soluble metabolites ~~
rnin
5 15 30 60 60 (no cells)
60.0 + 3.0 35.8 k 0.4 26.0 + 0.6 15.1 2 1.7 91.7 f 1.9
2.3 k 0.03 2.6 8.5 f 0.03 2.4 + 0.1 2.0 f 0.21 2.5 0.06
3.7 f 0.03 f 1.0 10.7 +. 2.1 16.5 f 4.5 0.9 0.15
+
~
33.3 f 2.7 52.0 f 1.2 59.7 -C 1.5 66.0 k 4.7 3.9 f 1.6
Metabolism of 1 -Acyl-2-acetyl-GPC
6932 TABLE I1
Water-soluble metabolites derived from catabolism of l-acyl-2-acetyl-p4C]GPC Neutrophils (10' cells/tube) were incubated with 0.5 nmol of 1a~yl-2-acetyl-['~C]GPC. At various time points, the reactions were stopped, and thelipids were extracted (22). The aqueous extract was concentrated and analyzed by TLC on Silica Gel G plates developed in ethanol/water/ammonium hydroxide (48:70:7, v/v). These data are expressed as the percentageof radioactivity recovered in the aqueous phase after lipid extraction. These data are presented as the mean f S.E. of four experiments. % of water-soluble radioactivity
Time
l-Lyso 2-ace- Unidentified
Phosphocholine Choline
CPC
tyl-GPC
10.7 k 1.9 21.8 k 1.7 26.3k 0.9 22.5 f 1.3
63.3 f 6.6 36.5 4.0 18.0f 3.2 14.5 f 2.4
rn~t..l~p-
min
55.8 f 1.4 15 16.2 f 3.4 3024.5 f 4.3 60 26.8 f 2.3
2.6 f 0.6 7.4 f 1.2 16.3 f 1.7 24.0 f 2.9
+
15.0 f 4.8 16.8 f 5.1 12.4 k 3.0 9.7 f 1.9
TABLE I11 Rates of catabolism of 1-alkyl-2-acetyl-GPC and I-acyl-2-acetyl-GPC at different concentrations of the substrate incontrol and A23187stimulated neutrophils Neutrophils (10' cells/tube) were incubated in HBSS (1ml) with different concentrations of l-['H]alkyl-2-acetyl-GPCor l-['4C]acyl2-acetyl-GPC in the absence (control) or in the presence of A23187 (2 FM). After 15 min of incubation, the reactionwas stopped and the lipids were extracted by the method of Bligh and Dyer (22). The phospholipids were analyzed by TLC on Silica Gel Gplates developed in chloroform/methanol/acetic acid/water (50:25:8:4, v/v) and the radioactivity remaining in 1-alkyl-2-acetyl-GPC or 1-acyl-2-acetylGPC was measured by zonal scraping and liquid scintillation counting. These data are presented as the mean f S.E. of three experiments.
0
m c
ls -
0
1-acyl-2-acetyl-GPC neutral lipids If-diacyl-GPC l-aCyl-t-lyso-GPC
1-"
20 0
0
20
40
60
Time (min) FIG. 4. Time course of the formation of labeled compounds derived from the catabolism of l-['4C]acyl-2-acetyl-GPC in A23187-stimulated neutrophils. Neutrophils (lo' cells/tube) were incubated with 0.5 nmol of l-['4C]acyl-2-acetyl-GPC. In the stimulated samples,A23187 (2 p ~ was ) added simultaneously with 1["C]acyl-2-acetyl-GPC. The lipids were extracted and analyzed by TLC on Silica Gel Gplates developed in chloroform/methanol/acetic acid/water (5025:84, v/v). The formation of labeled metabolites was determined by zonal scraping andliquid scintillation counting. These data are expressed as the percentage of the total radioactivity recovered after lipid extraction and are presented as the mean f S.D. of two experiments.
Subsequentanalysis of metabolites derivedfrom l-["CC] acyl-2-acetyl-GPC in stimulated cells indicated that there was anincreasedformation of 1,2-['4C]diacyl-GPC(Fig. 4) as compared to unstimulated cells (Fig. 3A). The amount of labeled 1,2-diacyl-GPC in A23187-stimulated cells was larger than that of labeled neutral lipids at all time points after 5 min of incubation. Two likely explanations for the increase of label inPCduring cell activationare 1) an increased capacity for the deacetylation-reacylation pathway to participate in the catabolism of 1-acyl-2-acetyl-GPC in stimulated cells, and/or 2) stimulatedneutrophilssimultaneouslyinpmol of substrate catabolized/ crease their capacity to remove the long chain at the sn-1 15 min/lO' cells Conc. (nmol/ml) position of 1-acyl-2-acetyl-GPC along with an increased caControl A23187 pacity to transfer thatlong chain into PC. In order to deter1-Alkyl-2-acetyl-GPC mine which of these two possibilities was occurring during 36 f 5 52 f 3 0.1 cell activation, experiments were performed in which acti140 f 23 135 f 13 0.5 vated neutrophils were incubated with I-acyl-2-acetyl-GPC 190 f 20 200 f 38 1 labeled at the sn-3position of the molecule. If the deacetyla192 f 22 325 f 58 2.5 167 k 17 317 f 44 5 tion-reacylation capacity increased, an increase in label found in 1,2-diacyl-GPC would be expected. Alternatively, if the loss 1-Acyl-2-acetyl-GPC of the fatty acylmoiety at the sn-1 position is the major 56+ 1 52 f 1 2 0.1 pathway that is increased during cell activation, an increase 282 f 23 270 k 44 0.5 in radioactivity associated with thewater-soluble metabolites 390 f 89 560 f 55 1 1158 f 172 658 f 123 2.5 would be expected. The results shown in Table IV confirm 2083 f 335 900 f 202 5 that the rate of catabolism of 1-acyl-2-acetyl-GPC is higher in A23187-stimulated than in unstimulated cells. Moreover, crease in catabolic rate induced by A23187. In the case of 1- analysis of metabolites derived from catabolism of l-acyl-2of both 1,2acyl-2-acetyl-GPC the rateof catabolism was increased up to acetyl-[14C]GPC indicated that the formation of labeled water solublemetabolites were %fold by A23187. The increase inthe catabolic rate was dia~yl-['~C]GPC and evident at all concentrations of the substrate used in these increased in stimulated cells. Therefore, both the cleavage of experiments. The data in Table I11 also indicate that, at the the long chain at the sn-1 position (producingthe water higher concentrations of the two phospholipids, activated soluble metabolites) and the deacetylation-reacylation reacto increased neutrophils catabolized 1-acyl-2-acetyl-GPCat a rate four to tion (producing 1,2-dia~yl-['~C]GPC) appear be upon A23187-induced stimulation. seven times faster than thatof PAF. Catabolism of 1 -Acyl-2-acetyl-GPC in Subcellular Fractions Neutrophils stimulatedwith A23187 synthesized PAF(0.32 f 0.03 nmol/2 X lo7 cells; mean & S.E.; n = 5). Therefore, it of the HumanNeutrophil-Another group of experiments was performed to initiallyidentify an enzyme activity which might was of interesttoevaluatethe effect of PAF M ) on the catabolism of 1-acyl-2-acetyl-GPC. These experiments indi- be responsible for the removal of the long chain at the sn-1 cated that the catabolism of 1-acyl-2-acetyl-GPC was not position of 1-acyl-2-acetyl-GPCintheneutrophils.These enzyme an significantly affected by concentrations of PAF comparable experiments indicated that the neutrophils contain t o those produced in uitro (data not shown). In addition, theseactivity which will cleave the sn-1position of 1-acyl-2-acetylin Fig. 5, indicatethatthe data excluded the possibility that endogenous PAF may be GPC.Theresults,illustrated responsible for the increased catabolism of 1-acyl-2-acetyl- majority of this enzyme activity resides in the cytosolic fraction. In addition, thisenzyme is able toremove the long chain GPC in A23187-stimulated neutrophils. ~~
~~
6933
Metabolism of 1 -AcyE2-acetyl-GPC TABLEIV Effect of neutrophil actiuation on the catabolismof I -a~yl-2-acetyl-['~C]GPC Neutrophils (lo7 cells/tube) were incubated with 0.5 nmol of l-a~yl-2-acetyl-['~C]GPC in the absence (control) or in the presence of A23187 (2 p ~ ) At . different time points, the reactions were stopped, and the lipids were extracted by the method of Bligh and Dyer (22). The radioactivity was measured in both the organic and the aqueous phase. The metabolites contained in the organic phase were then separatedby TLC on Silica Gel Gplates developed in chloroform/methanol/acetic acid/water (50:25:8:4, v/v). The labeled metabolites were determined by as the percentageof the totalradioactivity zonal scraping andliquid scintillation counting. These data are expressed f S.E. of three recovered in both the organic and the aqueous phase. These data are presented as the mean experiments. % of total radioactivity
-. ume
1-Acvl-2-acetvl-GPC 1-Acyl-2-lyso-GPC metabolites Water-soluble 1,2-diacyl-GPC
min
Control 5 15 30 60
81 f 4.3 65 f 5.5 54 k 6.1 39 f 4.4
A23187 5 15 30 60
65 6.8 33 & 4.6 20 f 3.2 12 2.2
*
*
1.7 f 0.2 2.2 0.1 2.9 f 0.3 3.4 0.6
* *
1.9 -t 0.4 3.4 f 0.8 5.9 1.4 14 f 3.9
+
14 f 3.8 29 k 5.2 36 f 5.5 42 & 1.2
2.1 f 0.2 2.4 0.2 2.7 2 0.5 2.4 f 0.3
4.7 f 1.2 17 k 2.3 20 f 2.4 22 2.1
27 f 5.2 46 +. 4.0 57 2 2.3 62 f 4.7
+
*
acyl-2-acetyl-GPC in the human neutrophil. In addition to the deacetylation-reacylation reaction by which PAF is meC tabolized, 1-acyl-2-acetyl-GPCis metabolized primarily by 'i 40 removing the long chain at the sn-1 position. At present we M 30 have not isolated the enzyme responsible for the hydrolysis 0 20 of the ester linkage of 1-acyl-2-acetyl-GPC (lipase A,, acyl 10 hydrolase). However, experiments in this study indicate that it preferentially hydrolyzes the ester linkage since l-alkyl-20 acetyl-GPC is not significantly hydrolyzed at the sn-1 posiCytosol Pellet cytosolic fraction of theneutrophil FIG. 5. Hydrolysis of the acyl chain at the sn-1 position of tion.Inaddition,the contains an enzyme activity which may be responsible for the l-acyl-2-acetyl-GPC, l-acyl-2-lyso-GPC, and 1,a-diacylGPC by subcellular fractions of the human neutrophils. Sub- initial hydrolysis of 1-acyl-2-acetyl-GPC. This activity apcellular fractions were obtained from human neutrophil preparations pearstobedifferentfromthe classicalphospholipase A, as described under "Experimental Procedures." Two-hundred pl of enzymes (23,24) since it prefers substrates containing a short the cytosol and membrane fraction (pellet) were added to 800 ~1 of chain (acetate) or no fatty acyl chain at the sn-2 position. HBSS containing 1.2 mM Ca'+ and 2 nmol of l-['4C]acyl-2-acetylGPC, l-['4C]acyl-2-lyso-GPC, or l-[l4Cj2-diacyl-GPC complexed to The subsequent fateof 1-lyso-2-acetyl-GPC is the removal of HSA (final concentration, 1 mg/ml). The reaction mixture was in- the acetate group and/or the removal of the choline or phoscubated at 37 "C for 30 min. The reactions were terminated by the phocholine group. The long chain which is initially removed addition of methanol/chloroform (2:1, v/v). The lipids were extracted from themolecule remains asa free fatty acid for ashort time and separated by TLC on Silica Gel G plates developed in hexane/ and is thenrapidly incorporated intotriglycerides or PC. No ethyl ether/formic acid (90:60:6, v/v). The radioactivity migrating significant formation of 1-acyl-2-acetyl-glycerol or l-acyl-2with free fatty acids was determined by zonal scraping and liquid scintillation counting. Thesedataare expressed as picomoles of acetyl-phosphatidylglycerol was observed during catabolism of three separate of 1-acyl-2-acetyl-GPC in the neutrophils. Although a very substrate catabolized/minute and are representative experiments. small amountof radioactivity can be found in the diglyceride fraction during catabolism of l-['4C]acyl-2-acetyl-GPC residue at the sn-1position from 1-acyl-2-lyso-GPC. In con- (
