Daniel H. Albert+ and Fred Snyder8. From the Medical and ...... Wykle, R. L., and Snyder, F. (1976) in The Enzymes of Biological. Membranes (Martonosi, A., ed) ...
Biosynthesis of l-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (Plateletby activating Factor)from 1-Alkyl-2-acyl-sn-glycero-3-phosphocholine Rat Alveolar Macrophages PHOSPHOLIPASE AS AND ACETYLTRANSFERASE ACTIVITIES DURING PHAGOCYTOSIS AND IONOPHORE STIMULATION* (Received for publication, May 24, 1982)
Daniel H. Albert+ andFred Snyder8 From the Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830
l-Alkyl-2-acyl-sn-glycero-3-phosphocholine(alkyl- in phospholipid metabolism in the lung (for reviews, see Refs. acyl-GPC) comprises 11%of the total phospholipids of 1 and 2). One potentially important aspect of their role may rat alveolar macrophages. This endogenous pool of be the biosynthesis and/or catabolism of inflammatory mealkylacyl-GPC was prelabeled by incubating the mac- diators derived from phospholipids. As part of our study of rophages with [1,2-3H]alkyllyso-GPC (54 Ci/mmol), pulmonary phospholipid metabolism, we have investigated which enters the cells and is acylated. The effect of the biosynthesis and cellular release of alkylacetyl-GPC,’ i.e. various stimuli on the synthesis and release into the platelet-activating factor. media of labeled alkylacetyl-GPC (platelet-activating Alkylacetyl-GPC is a biologically active phospholipid with factor) from thecells was used to establish the role of potent platelet-activating and antihypertensive activities (for inactive alkylacyl-GPC as a precursor of the biologi- reviews, see Refs. 3-5). Ithas also beenimplicated as a cally active derivative. A phagocytic agent (zymosan, mediator in immune responses including inflammation and 100 pg/ml) and an ionophore (A23187,2 PM) stimulated anaphylaxis (6).A number of investigators have demonstrated the releaseof both alkylacetyl-GPC and alkyllyso-GPC into themedia at the expense of cellular alkylacyl-GPC. that alkylacetyl-GPC is released by a variety of tissues and Phospholipase Az activity (at pH 4.5 and in 1mM EDTA) cell types, e.g. basophils (7),platelets (8),neutrophils (91, and was also increased in the media. The stimulatory effect macrophages (10-12).Specific enzymatic reactions involved of zymosan and the ionophore on alkylacetyl-GPC re- in the biosynthesis of alkylacetyl-GPC have been documented lease was prevented by mepacrine (0.1 mM), an agent in some cell types. These reactions include the formation of that inhibits the release of fatty acids from phospholip- the etherbond by alkyldihydroxyacetone-P synthase(13, 14), by ids. These data indicate that phospholipase activity is transfer of phosphocholine to 1-alkyl-2-acetyl-sn-glycerol required for the biosynthesis of alkylacetyl-GPC.How- a cholinephosphotransferase (14,15), and acetylation of alever, since the inhibitory effect of mepacrine was not kyllyso-GPC by an acetyltransferase (15-19). It is notknown apparent when acetate was present, it appears that thewhich of these reactions is quantitatively the most important acetylation step is rate limiting. Exposure of alveolar in the biosynthetic pathway(s) leading to the synthesis of macrophages in culture to zymosan or A23187 stimu- alkylacetyl-GPC in macrophages or any other cell. However, lated acetyltransferase activity 250-300%. In contrast, it is clear that acetyltransferase is markedly stimulated by , stimulated agents known to evoke platelet-activating factor responses phorbol myristate acetate (1.6 p ~ ) which the accumulation of lysophospholipids but not the level (18, 19). of alkylacetyl-GPC in the media, did not substantially In theory, the stimulation of the release of alkylacetyl-GPC increase acetyltransferase activity. We conclude that could be due to a combined enzymatic process: 1) activation alkylacyl-GPC serves as a precursorof alkylacetyl-GPC of phospholipase A2, which causes an increase in the concenand that the production of this potent mediatorby rat tration of the precursor alkyllyso-GPC; and 2) stimulation of alveolar macrophages can be stimulated by agents that affect phospholipase Az and acetyltransferase activi- acetyltransferase activity, which catalyzes the acetylation of the lyso precursor. Ourapproach to investigate this hypothesis ties. The latter enzyme appears to have a regulatory for the biosynthesis of alkylacetyl-GPC hasbeen to prelabel function in the biosynthesis of alkylacetyl-GPC. the endogenous pool of alkylacyl-GPC by incubating macrophages with1-[1’,2’-’HH]alkyllyso-GPC of high specific activity, and then to determine theeffect of various stimuli or inhibiAlveolar macrophages appear to have a major responsibility tors on the synthesis andrelease of alkylacetyl-GPC into the * This work was supported by the Office of Health and Environ- media. We demonstrate here that alkylacyl-GPC is an effecmental Research, United States Departmentof Energy Contract DE- tive precursor of the acetylated bioactive phospholipid and that stimulationof rat alveolar macrophages by zymosan and AC05-760R00033), National Heart, Blood, and Lung Institute Grant HL-27109-02,and American Cancer SocietyGrant BC-70M. A prelim- the ionophore A23187 increases the release of alkylacetylinary report of this work was presented at the 66th Annual Meeting GPC and alkyllyso-GPC (derived from alkylacyl-GPC) from of the Federation of American Societies for Experimental Biology, the alveolar macrophages. The release is correlated with an April 18,1982, New Orleans, LA. The costs of publication of this increase in intracellular acetyltransferase and an increase in article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of an Andrew W. Mellon Foundation Fellowship. 3 To whom correspondence should be addressed.
’
The abbreviationsused are: alkylacetyl-GPC, I-alkyl-2-acetyl-snglycero-3-phosphocholine; TPA, 12-0-tetradecanoylphorbol-13-acetate; BSA, bovine serum albumin.
+
97
98
Biosynthesis
of Alkylacetyl-GPC
extracellular phospholipase AZ activity. It is equally significant that mepacrine, a phospholipase AZ inhibitor, can effectively reduce these responses and that acetate can reverse this inhibition. EXPERIMENTAL
PROCEDURES
Materials-1-[1’,2’-,‘H]Alkyllyso-GPC (54 Ci/mmol) was prepared from 1-[1’,2’-“Hlalkylacetyl-GPC by deacylation with the monomethylamine reagent described by Clarke and Dawson (20). The preparation of [“Hlalkylacetyl-GPC from choline plasmalogens of beef heart has previously been described (21). [l-‘4C]Acetyl-CoA (5.55 mCi/mmol) was purchased from Amersham Corp. Zymosan, ionophore A23187, quinacrine dihydrochloride (mepacrine), 4-bromophenacyl bromide, and TPA were obtained from Sigma Chemical Co. Hexadecyllyso-GPC was prepared by phospholipase A, treatment of rat-1-hexadecyl-2-cis-9’.octadecenoyl-GPC (16). Preparation of Alveolar Mucrophages and Culture ConditionsThe lungs of 4-month-old CDF rats were lavaged with 50 ml (per rat) of fluid (10 ml/wash at 37 “C) comprised of RPM1 1640 containing 10% fetal calf serum, 12 mM lidocaine by following the procedure described by Holt (22). The pooled lavage fluids were centrifuged at room temperature for 7 min at 160 x g and the resulting cell pellet was resuspended in RPM1 1640. The cell suspension was then plated into 35-mm plastic Petri dishes and incubated at 37 “C in 5% CO? for 2 h. We removed nonadherent cells from the adherent macrophages by washing the monolayer vigorously with RPMI. Generally, the lavage fluid from one rat provided sufficient cells for four Petri dishes (50-100 pg of protein/dish). Cells were maintained for 2-8 h in culture in RPM1 media containing 1 mg/ml BSA (BSA/RPMI media) and the indicated additions. To terminate the experiments, the media were collected, centrifuged at 4 “C to remove any cells, and then extracted by the method of Bligh and Dyer (23), except that the methanol contained 2% glacial acetic acid. In some instances, aliquots of the media were removed before extraction for assay of phospholipase AL or acid phosphatase activity. The cells adherent to the Petri dish were washed thoroughly with phosphate-buffered saline at 4 “C! and then collected with a rubber policeman in 1 ml of phosphatebuffered saline. Aliquots were taken for enzyme assays and protein determination (24), and the remaining suspension was extracted of lipids with chloroform/methanol (23). Analysis of Radioactive Lipids-For routine analysis, aliquots of the lipid extracts obtained from the media and cells, along with appropriate standards, were applied on thin layer chromatography plates coated with 250~pm thick layers of Silica Gel G. The plates were then developed in chloroform/methanoi/glacial acetic acid/water (50:25:8:4, v/v). The proportion of methanol and water in the developing solvent was often increased by as much as 20 and 50%, respectively, depending on relative humidity to ensure that alkylacetyl-GPC (RF = 0.37) was effectively separated from other closely related labeled phospholipids including alkyllyso-GPC (RF = 0.24) and alkylacyl-GPC (RF = 0.75). After development, the plates were exposed to Ir vapor to locate the positions of the various phospholipid classes. The silica gel layer was then collected in 2-mm fractions using a zonal scanning technique (25) and the radioactivity was determined in each fraction. In initial experiments to verify the identity of the radioactive metabolite which co-chromatographed with alkylacetylGPC, pooled fractions of this product were treated with phospholipase C (26). The resulting labeled nonpolar lipid derived by phospholipase C treatment co-chromatographed with the expected product, alkylacetylglycerol, on a silica gel layer developed with chloroform/methanol (982, v/v). The ether-linked lipid content of choline and ethanolamine glycerophospholipids was determined by the method described by Clarke and Dawson (20). Enzyme Assays-Phospholipase A, was assayed under the two sets of optimal conditions described by Wightman et al. (27), except that the substrate was a choline glycerophospholipid fraction extracted from rat alveolar macrophages that had incorporated and acylated l[1’,2’-“H]alkyllyso-GPC. This substrate consisted of 35% alkglacylGPC (24,000 dpm/nmol), 65% diacyl-GPC (not labeled). Aliquots of media or sonicated cell suspensions (100 pg of protein) were incubated for 15 h at 37 “C in a total volume of 150~1 with 2 nmol of phospholipid substrate and either 0.1 M potassium acetate, 1 mM EDTA (pH 4.5) or 0.1 M Tris-HCl, 2 mM CaCIL (pH 8.5). Incubation media were extracted and the radioactive phospholipids were analyzed by thin layer chromatography as described above. The amount of radioactivity recovered in the lysocholine glycerophospholipid fraction was used to calculate the enzymatic activity. Acid phosphatase was assayed
using 4-nitrophenyl phosphate as the substrate by the method described by Schnyder and Baggiolini (28). Acetyltransferase was assayed by the method previously described by Wykle et al. (16). Sonicates of pooled cell suspensions were prepared in a 0.25 M sucrose, 1 mM dithiothreitol solution. Aliquots (700 ~1 containing 80-150 pg of protein) were incubated for 15 min at 37 “C in a final volume of 1 ml with 30 nmol of hexadecyllyso-GPC, 100 nmol of [“Hlacetyl-CoA (4000 dpm/nmol), and 0.1 mmol of TrisHCl (pH 6.9). The incubation mixture was extracted and the radioactive products were analyzed by thin layer chromatography. In one experiment, [JH]alkyllyso-GPC was included and [‘Y!]acetyl-CoA was used in place of [“Hlacetyl-CoA. Thin layer chromatography of the products showed that the major ,‘H- and ‘%-labeled lipid co-chromatographed and had a mobility identical with that of a standard of alkylacetyl-GPC. Student’s t test was used to calculate probabilities of significance. RESULTS
Phospholipid Composition-The phospholipid composition of rat alveolar macrophages is given in Table I. The aikylacyl-GPC fraction represents a substantial proportion (>lO%) of the total phospholipids of these cells and was readily labeled by incubating macrophages with [ 1,2-“HIalkyllyso-GPC (Fig. 1). After a 4-h incubation, greater than 80% of the radioactivity recovered in the cells was in the alkylacylGPC fraction, less than 5% was in the alkyllyso-GPC fraction, and only traces of radioactivity (tO.l%) were in the alkylaceTABLE I Phospholipid composition of rat alveolar macrophages The total phospholipid content of cells was 0.52 + 0.03 nmol/wg of protein. Results are the average of two determinations except that the value for alkylacyl-GPC represents the mean of four measurements (SE. = kO.89). ND = not detected. Total Phospholipid class phosphorus r+
Diacyl-GPC Alkylacyl-GPC Alk-I-enylacyl-GPC Diacyl-GPE Alkylacyl-GPE Alk-l-enylacyl-GPE Phosphatidylserine Sphingomyelin Other (solvent
+ phosphatidylinositol front
and origin)
21.5 11.7 ND 14.4 0.2 12.0 14.2 13.7 12.2
FIG. 1. Time course for the incorporation of radioactivity into alkylacyl-GPC of rat alveolar macrophages. Cells were incubated for the indicated time in 1 ml of BSA/RPMI media containing 0.03 nmol of [1,2-“H]alkyllyso-GPC (1.7 pCi/ml). The cells were then harvested, the lipids were extracted, and the radioactivity in phospholipids was determined as described under “Experimental Procedures.” Results are representative of five experiments. Shown are the means of duplicate measurements from one experiment. SE values are indicated by the vertical line.
Biosynthesis of Alkylacetyl-GPC
99
substantially, so that aftera 1.5-h incubation, cells exposed to either stimuli had 8-15% less total radioactivity than did the These control cells (Fig. 2 A ) . This loss of cellular radioactivity was reflected primarily by a -20% reduction in the radioactivity in the alkylacyl-GPC fraction of phospholipids of cells exposed to the stimulifor 1.5 h (Fig. 2B). In contrast, the rate of loss of radioactivity from the intracellularalkyllyso-GPC fraction was approximately one-fourth that of the alkylacyl-GPC fraction andwas not appreciably alteredby exposures to zymosan or the ionophore. Thus, the radioactivity released from the stimulated macrophages into themedia was derived fromthe cellular pool of alkylacyl-GPC. Chromatographic analysis of the radioactivephospholipids released into the media demonstrated that ["H]alkylacetylGPC accumulated in themedia of cells exposed to ionophore and at a slower rate and to a lesser extent than in the media of cells exposed to zymosan (4.0 and 2.5%, respectively, of the I 1 I I B A radioactivity in the media at60 min, Fig. 3A). Ionophore and zymosan also stimulated the release of alkyllyso-GPC, the predominant labeled phospholipid in the media (67 and 9076, respectively, of the total 60 at min, Fig. 3 B ) .Phorbol myristate 800 acetate, in contrast to the ionophore and zymosan, had no effect on the quantity of ["Hlalkylacetyl-GPC found in the i media (0.5% of the total, Fig. 3A), although it did stimulate E, 700 the accumulation of alkyllyso-GPC (93%of the total "H, Fig. 0 z 3B). .E 600 Significant quantities of alkylacyl-GPC (29% of the total at .$ 60 min) were also detected in the media of cells exposed to 1 200 the ionophore(Fig. 3C). Thereason for the appearanceof this labeled alkylacyl phospholipid in the media is not clear. It 70 apparently is not due to cell lysis since exposure of macroi phages to A23187 did not affect the release of lactate dehydrogenase oracetyltransferaseintothemedia(datanot 0 I I I 0 1 2 3 shown). Houn Phospholipase AzActiuity-The decrease in label fromthe FIG. 2 . Effect of zymosan and ionophore on the loss of intraintracellular alkylacyl-GPC pool from cells exposed to zymocellular radioactivity from alveolar macrophages. Cellular alsan or ionophore and the accumulation of labeled alkyllysokylacyl-GPC was labeled by incubating macrophages with ['HlalkylGPC in the mediawould appear to be the result of a stimulalyso-GPC for 4 h. After washing the cells with RPMI, themedia was then replaced with BSA/RPMI media (control, O ) , or BSA/RPMI tion of phospholipase A2 by these stimuli.Addition of a , media containing either zymosan (200 pg/ml, A) or A23187 (2 p ~ 0) phospholipase A2 inhibitor, mepacrine, to the incubation mefor the indicated time. Cells were then harvested and radioactivity dia of cells exposed to zymosan or the ionophore prevented was determined: A , per cent of total radioactivity incells (5092 k 576 the loss of radioactivity from the cellular pool of alkylacyldpm/mg protein at time0) and B , radioactivity in the alkyllyso-GPC GPC(Table 11). Furthermore,theinhibitor also causeda (broken lines) and alkylacyl-GPC (solid lines) fractions of the cells. sharpreduction (60%) in theamount of alkylacetyl-GPC Plotted values are the means from two experiments withS.E. value accumulated in the media of cells exposed to the ionophore indicated by the vertical line.
tyl-GPC fraction. The remaining 11-15% of radioactivity recovered inother fractions was not further characterized. results demonstrate that alveolar macrophages have an efficient mechanism for the uptake and acylation of the alkyl lysophospholipid for storage as an inactive precursor (alkylacyl-GPC) of the bioactive phospholipid (alkylacetyl-GPC). Cellular Metabolismof Alkylacyl-GPC-Exposure of macrophages labeled with ['H]alkylacyl-GPC to aphagocytic agent (zymosan) or a calcium ionophore (A23187) led to an increase in the appearance of labeled metabolites in the media and a commensurate loss of radioactivity from thecells (Fig. 2 A ) . At the beginning, the rate of loss of radioactivity from the cells was highest in cells exposed to the ionophore. However, after an initiallag of approximately l h, the rateof loss of labelfrom the cells exposed to zymosan also increased
: D
T
'I
6o
0
20
40 Minutes
Alkyllyso-GPC
3oy
60 Minutes
Minutes
FIG. 3. Time course for the accumulation of radioactive metabolites in the media of zymosan- and ionophore-stimulated macrophages. Cellular alkylacyl-GPC was prelabeled as described in Fig. 2. The macrophages were then incubated with BSA/RPMI media containing the following additions: none ,).( zymosan (200 pg/ml, A), ionophore A23187 (2 p ~O), , or TPA (1.6 p ~ 0). , After the indicated time, radioactive phospholipids in the mediawereanalyzed as described under"Experimental Procedures." Resultsarethemeans from three experiments with S.E. values indicated by the uertical line. Probabilities of significant differences compared to control were calculated for the 60-min interval values. p < 0.05 indicated by asterisk.
100
Biosynthesis of Alkylacetyl-GPC
and toa lesser extent (50%)in cells exposed to zymosan (Table 11).Bromophenacyl bromide(0.1 mM), another phospholipase inhibitor, also had a similar effect on the conversion of alkylacyl-GPC to alkylacetyl-GPC (data not shown). The agents that stimulated the release of alkyllyso-GPC from cells were also tested for their effect on phospholipase AS activity. The A:! activity when assayed at pH 4.5 with 1 mM EDTA in the incubations was approximately 1.4- to 1.9fold higher in the media from the cells exposed to zymosan, ionophore, or TPA thanin the media from cells not exposed to the stimuli (Table 111).However, the total activity of the cells and media combined was not significantly increased by the stimuli. We interpret these results to indicate that such agents increase the amount of phospholipase released from the cells intothemediaratherthandirectlystimulating phospholipase ABactivities. The activit.y of a phospholipase A2 (27) measured a t neutral pH in the presence of calcium in either the media or cells was not substantially altered by the stimuli (Table 111). In order to gain insight on whether the phospholipase A:, activity affected by zymosan, TPA, or the ionophore was of lysosomal origin, we also examined the effect of these stimuli on therelease to the mediaof acid phosphatase, anenzyme of lysosomal origin (28). The total amountof phosphatase activity recovered was not significantly altered by the zymosan or ionophore treatment. Between 82 and 94% of the total phosphatase activity was intracellular (Table IV). However, after exposure of the cells to zymosan or theionophore, a significant
increase occurred in the relatively small amount of phosphatase activity released into the media (TableIV). Thus, these agents appear to stimulate therelease of lysosomal enzymes from the macrophages. TPA caused a moderate stimulation (3540%)of phosphatase activity in both the cells and media, which suggests that TPA can activate acid phosphatase as well as affect its release from macrophages. Influence of Acetate on Mepacrine Inhibition of the Zymosan- and Ionophore-stimulated Release of AlkylacetylGPC-The effect of acetate on the accumulation of alkylacetyl-GPC in the mediaof cells exposed to stimuli and mepacrine (Table V) provides indirect evidence that indicates the important role of the acetyltransferase-catalyzed step in the TABLE IV Effect of zymosan, ionophore A23187, a n d TPA on acid phosphatase activity in alveolar macrophages Cells were incubated for 2 h in BSA/RPMI mediacontaining zymosan, A23187, or TPA as described in Table 111. Acid phosphatase was assayed in the media and cells as described under “Experimental Procedures.” Values in parentheses refer to per cent control, Results represent the meanf S.E. of four assays. Activity Stimulus Media
None (control) Zymosan A23187 TPA
Effect of zymosan, ionophore A23187, a n d mepacrine on intracellular alkylacyl-GPC and the release of alkylacetyl-GPC from alveolar macrophages Cells prelabeled as described in Fig. 2 were incubated for 2 h with BSA/RPMI media or BSA/RPMI media containing either zymosan (200 pg/ml) or A23187 (2 PM), with or without mepacrine (0.1 mM). Radioactive alkylacetyl-GPC in the media and intracellularalkylacylGPC was determined as described under “Experimental Procedures.” Results are expressed as the mean f S.E. Control values: alkylacylGPC ( n = 4), 2684 f 116 dpm/pg of protein; alkylacetyl-GPC ( n = 6), 2848 + 1013 dpm/mg of protein. Cellular alkylacylAlkylacetyl-GPC GPC
146 158 152 200
TABLEV Effect of acetate on alkylacetyl-GPC release from alveolar macrophages Cells were prelabeled and incubatedas described in TableI1 except sodium acetate (0.2 m) was included in the incubation media as indicated. Results are the mean f S.E. Values in parentheses refer to per cent control. Alkvlacetvl-GPC in media Stimulus
N
Minus Plus mepacrine mepacrine
c
PIUS ~
Plus acetate acetate ~ ~ and ~mepacrine,, f ~
dpm/ml
media
Minus mepacrine % control
104 f 21 75 f 7 99 f 3“ 75 f 5 107 f 18”
~
in
Stimulus
100
Total
“ p < 0.001 when compared to control value. *p< 0.05 when compared to control value.
TABLE I1
None (control) Zymosan A23187
Cells
pmol/h 8.9 f 0.8 (100) 137 f 21 (100) 11.9 f 1.0“ (133) 146 f 12 (106) 27.6 f 1.3”(308) 124 f 16 (90) 11.6 f 1.3b (133) 188 f 7“ (136)
None ( n = 3) Plus, mepacrlne
Zymosan (n = 2)
204 -c 62 (118 f 32) 321 f 98 (127 -+ 8) 1615 -+ 517 (146 -t 50) 188 -+ 12 (101 f 24)
178 f 60 (100) 259 f 93 (100)
102 -+ 36 167 c 35 85 f 52” 505 k 224206 k 105“ 100
‘*p< 0.05 when compared to the values obtained for the zymosan or A23187 samples minus mepacrine.
1121 f 58 (100) 194 f 34
A23187 ( n = 3)
TPA (n = 2)
(100)
255 f 132 (168 f 96) 366 k 182 (142 f 22) 1763 f 801 (159 f 74) 123 f 28 (63 ? 3)
‘‘ See Table I1 for the inhibitory effects of mepacrine on stimulus.
TABLE I11 Effect of various stimulion phospholipase Az activity in alveolar macrophages Cells were incubated for 2 h in BSA/RPMI media containing no additions, zymosan (200 pg/ml), A23187 (2 p ~ ) or , TPA (1.6 PM). The media and cells were then collected and phospholipase A:, activity was assayed as described under “Experimental Procedures.” Values in parentheses are activities of control (picomoles/flask/h). Results are expressed as the mean f S.E. of two experiments. Assay conditions for activity pH 4 . 5 , l mM pH EDTA
Stimulus Media
Cells
8.5, 2 m~ CaCL
Cells plus media
Cells plus media
Media
%>control
None
100
Zymosan A23187 TPA
(13.4 k 1.1) 144 f 7 187 f 6 136 f 6 ~~~~
100 (44.3 f 1.0) 72 f 2 93 f l 89 f 2
100
100
100
100
(58) 88 114 100
(17.8 f 1.0) 100 2 2 118 f 1 106 f 3
(16.4 f 0.3) 96 f 5 108 f 61 111 k 5
(34)
”~
..
100
115 109 -
~
Biosynthesis of Alkylacetyl-GPC TABLEVI ionophore- and zymosan-mediated stimulation of acetyltransferase Alveolar macrophages were incubated for 1h in BSNRPMI media. The cells were then harvested and subjected to sonication. Acetyltransferase in cell homogenates was assayed as described under “Experimental Procedures.” Results are the mean +- S.E. of three experiments. Additions
activity Specific pmollmgproteinl
% control
man
186 f 18 None (control) 442 & 121“ Zymosan (200 pg/ml) 465 rt_ 124“ Ionophore A23187 (2 pM) 231 132f 29 TPA (1.6 p ~ ) “ p < 0.02 when compared to control value.
100 238 252
TABLEVI1 Ionophore-mediated stimulation of acetyltransferase activity in sonicated preparations of alveolar macrophages Komogenates o f cells were prepared and assayed for acetyltransferase activity with the indicated additions as described under “Experimental Procedures.” Values represent the mean -C S.E. of three experiments. Specific activity
Additions Minus A23187
None (control) 1mM EDTA
I mM CaC12
Plus A23187
pmollmg protein/min 580 f 163“ 238 & 84 153 f 49 18 2 10’ 275 & 94 309 & 94
“ p < 0.05 when compared to control value minus the A23187.
biosynthesis of the bioactive lipid. Quantitative assessmentof the effectby acetate on the accumulationof alkylacetyl-GPC was hampered by a large variation in the magnitude of the responses in different cell populations. However, certain consistent features of these responses to acetate were apparent. Addition of acetatealone wasineffectivein stimulating alkylacetyl-GPC accumulation and had only slight effect on the responses in the ionophore- or zymosan-treated cells (Table V). However, the presence of acetate in the media prevented the inhibitory effects of mepacrine on the release of alkylacetyl-GPC from macrophages after stimulation by zymosan and A23187 (Table V). The extent that alkylacetylGPC accumulated in the mediaof cells exposed to TPA (with and without mepacrine)was not increased by acetate (Table V) . Acetyltransferase Actiuity-Homogenates prepared from cells that had been incubated for 1 h in media containing zymosan or thecaIcium ionophore contained a higher level of acetyltransferase activity than homogenates from cells not VI); TPA was relatively ineffecexposed to the stimuli (Table tive in affecting acetyltransferase activity. In addition to the stimulatory effect of ionophore A23187 on intact cells, the ionophore also directly stimulated acetyltransferase activity in cell homogenates when the assay was done in the absence of added calcium (Table VII). The activityin the homogenate was inhibited by EDTA, whereas the activity was not stimulated above controlvalues by the addition of calcium (Table VII) . DISCUSSION
101
GPC released from the cells stimulated with zymosan or the ionophore (Fig. 4).The effects of metabolic inhibitors further strengthen theproposed precursor roleof the inactive form of the ether phospholipid, alkylacyl-GPC, and the role of phospholipase A2 in the formation of the important lysophospholipid that serves as the substrate for the acetyltransferase in the synthesisof alkylacetyl-GPC. Mepacrine and bromophenacyl bromide prevented both the decreasein radioactivity in cellular alkylacyl-GPC and the accumulation of radiolabeled alkylacetyl-GPC in the media associated with stimulation of macrophages. It is recognized that mepacrine and bromophenacyl bromide may have other actions that could affect the cellular metabolism of phospholipids. These include inhibition of phospholipase C (30) and the capacityof mepacrine toformderivatives with phosphatidylethanolamine (31). However, the ability of these inhibitors to prevent the decrease in the labeled pool of cellular alkylacyl-GPC associated with the accumulationof alkyllyso-GPC, the expected product of a deacylationreaction, is consistentwith the ability of mepacrine and bromophenacyl bromide to inhibit fatty acid release via phospholipase A2 in intact cells (30, 32, 33). In macrophages, at least two types of phospholipase A2 activities have been described that utilize diacyl-GPC as a substrate: one has a pH optimum of 4.5, does not require added calcium, and is lysosomal; the other hasa pH optimum of 8.5, requires added calcium, and is microsomal (27, 34, 35). Inthepresent investigation when assayed underoptimal conditionswith 1,2-[”H]alkylacyl-GPC as a substrate, both types of phospholipase AZ activities were also detected, although at a substantially lower level than reported for the deacylation of diacyl-GPC labeled with arachidonate at the sn-2 position (27).It is possible that the phospholipase A2that utilizes alkylphospholipids as substrates is not the same enzyme that hydrolyzes diacyl-GPC. However, the phospholipase AS activity associated with the alkyllyso-GPC release from macrophagesdoes appear to beof lysosomal origin since of [“Hlalkyllyso-GPC agents that stimulated the accumulation and phospholipase Az in the mediaalso caused a corresponding increase in acid phosphatase activity. The A2 activity released into the media had characteristics similar to those reported for lysosomal phospholipase A2 (27,34,37). Furthermore, the stimulatedrelease of a phospholipase A:! and of the lysosomal enzyme acidphosphatase into the media from cells exposed to zymosan reflectsthe abiIity of inflammatory agents to stimulate the release of lysosomal enzymes from macrophages (36). Nevertheless, we cannot discount the possibility that other nonlysosomal phospholipases not measurable under our assayconditions are also affected by the stimuli. Clearly, if alkylacetyl-GPC is derived from alkylacyl-GPC as our results indicate, acetylation of the lysophospholipid intermediate is a necessary step and may represent an important regulatory point in the synthesisof the biologically active phospholipid. Wykle et al. (16) have reported the presence of a specific acetyltransferasein a variety of tissues that is ALKYLACYL-GPC
I
PHOSPHOLIPASE A 2
Alveolar macrophages form and release alkylacetyl-GPC ALKYLLYSO-GPC and alkyllyso-GPC when exposed to phagocytic or ionophoretic stimuli (Ref.29 and present study).We interpret that the ACETYL-CoA accumulation of the labeled alkyl lysophospholipid found in ACETYLTRANSFERASE the media at theexpense of labeled cellular alkylacyl-GPC is due to an increase in the activity of a phospholipase A2. This ALKYLACETYL-GPC enzyme appears tobe closely coupled with the acetyltransferFIG. 4. Proposedpathway for thebiosynthesis ase activity that is responsible forthe formationof alkylacetylacetyl-GPC by rat alveolar macrophages.
I
of alkyl-
102
Biosynthesis ofAlkylacety1-GPC
6. McManus, L. M., Morley, C. A., Levine, S. P., and Pinckard, R. capable of catalyzing the acetylation of alkyllyso-GPC. This N. (1979) J. Immunol. 123,2835-2&1 activity has also been recently demonstrated in murine peri7. Hanahan, D. J., Demopoulos, C. A,, Liehr, J., and Pinckard, H. toneal macrophages (18), and in polymorphonuclear leukoN. (1980) J. Biol. Chem. 255,5514-5516 cytes (15, 19) and eosinophils from humans (19). Moreover, 8. Chignard, M., Le Couedic, J. P., Vargaftig, B. B., and Benveniste, acetyltransferase activity has been shown to be modulated by J. (1980) Br. J. Haematol. 46, 455-464 calcium ionophore A23187 (19) and zymosan (15, 18). The 9. Lynch, J . M., Lotner, G. Z., Betz, S. J., and Henson, P. M. (1979) J. Immunol. 123, 1219-1226 mechanismfor the in vivo regulation of acetyltransferase activities in rat alveolar macrophages, as well as other cells, 10 Mencia-Huerta, J. M., and Benveniste, J. (1981) Cell. Immunol. 57,281-292 has yet to be established. The stimulationof acetyltransferase 11. Camussi, G . , Tetta, C., Bussolino, F., Masera, C., Emanuelli, G., by a calcium ionophore in intact cells and the inhibitionof its Ragni, R., and Porcellini, G. (1980) Panminerva Med. 22, 117activity in homogenates by acalcium chelatingagentas 124 observed in the present study suggests a regulatory function 12. Mencia-Huerta, J. M., and Benveniste, J . (1979)Eur. J. Zmmunol. 9,409-415 of calcium. On theotherhand,ourobservationthatthe 13. Wykle, R. L., and Snyder,F. (1976) in The Enzymesof Biological ionophore and not calciumper se stimulated acetyltransferase Membranes (Martonosi, A., ed) Vol. 2, pp. 87-117, Plenum in homogenates suggests that the ionophore can assert a direct Press, New York effect on the enzyme rather than just oncalcium availability. 14. Renooij, W., and Snyder, F. (1981) Biochim. Biophys. Acta 663, The regulatory significance of the acetylation reaction in 545-556 alveolar macrophages is illustrated by the effect of acetate 15. Alonso, F., Gil, M. G., Sanchez-Crespo, M., and Mato,J. M. (1982) J. Biol. Chem. 257, 3376-3378 upon the stimulation of the formation and cellular release of 16. Wykle, R. L., Malone, B., and Snyder, F. (1980) J. Biol. Chem. alkylacetyl-GPC by ionophore A23187 under conditionsin which deacylation is partially inhibited by mepacrine. The 17. 255, 10256-10260 Mueller, H. W., O’Flaherty, J. T., and Wykle, R. L. (1982) Lipids fact that, when acetate is added to the incubation media, the 17, 72-77 inhibitory effect of mepacrine on the formation of alkylacetyl- 18. Ninio, E., Mencia-Huerta, J. M., Heymans, F., and Benveniste, J . (1982) Biochim. Biophys. Acta 710, 23-37 GPC is prevented suggests that the rate-limiting stepin t,he 19. Lee, T., Malone, B., Wasserman, S. I., Fitzgerald, V., and Snyder, formation of the bioactive phospholipid from alkylacyl-GPC F. (1982) Biochem. Biophys. Res. Commun. 105, 1303-1308 is the acetylation of alkyllyso-GPC rather than the deacyl20. Clarke, N. G., and Dawson, R. M. C. (1981)Biochem. J. 195,301ation of alkylacyl-GPC. Indeed, an increased formation of 306 alkyllyso-GPC without a parallel increase in acetyltransferase 21. Blank, M. L., Lee, T., Fitzgerald, V., and Snyder, F.(1981)J. Biol. activity may not be sufficient to stimulate alkylacetyl-GPC Chem. 256, 175-178 formation. This is apparent with TPA, a membrane pertur- 22. Holt, P. (1979) J. Immunol. Methods 27, 189-198 bant thatincreases the level of the lysophospholipid precursor, 23. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917 but unlike the zymosan or ionophore does not appreciably 24. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 stimulate acetyltransferase and, therefore, does not induce an 25. Snyder, F., and Smith, D. (1966) Sep. Sci. 1, 709-722 accumulation of alkylacetyl-GPC in the media even in the 26. Mavis, R. D., Bell, R. M., and Vagelos, P. R. (1972) J. Biol. Chem. presence of added acetate. 247,2835-2841 We conclude that in rat alveolar macrophages both phos- 27. Wightman, P. D., Dahlgren, M. E., Davies, P., and Bonney, H . J. (1981) Biochem. J . 200,441-444 pholipase Ar and acetyltransferase behave as synergistic en28. Schnyder, J., and Baggiolini, M. (1978) J. Exp. Med. 148, 435zymes in the synthesis of alkylacetyl-GPC fromalkylacyl450 GPC, with alkyllyso-GPC as an intermediate (Fig. 4). The 29. Arnoux, B., Duval, D., and Benveniste, J. (1980) Eur. J . Clin. acetyltransferase activity would appear to be an important Znuest. 10,437-441 regulatory enzyme in the biosynthesis of platelet-activahg 30. Hofmann, S.L., Prescott, S. M., and Majerus, P. W. (1982)Arch. Biochem. Biophys. 215,237-244 factor.
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31. Dise, C. A,, Burch, J. W., and Goodman, I). B. (1982) J . Biol. Chem. 257,4701-4704 32. Lapetina, E. G., Billah, M. M., and Cuatrecasas, P. (1981) J . B i d . Chem. 256,5037-5040 33. Vargaftig, B. B. (1977) J . Pharm. Pharmacol. 29,222-228 34. Franson, R. C., and Waite, M. (1973) J. Cell Biol. 56,621-627 35. Wightman, P. D., Humes, J. L., Davies, P., and Bonney, H . J. (1981) Biochem. J . 195, 427-433 36. Hsueh, W., Kuhn, C., 111, and Needleman, P. (1979) Biochem. J. 184, 345-354 37. Franson, R., Beckerdite, S., Wang, P., Waite, M., and Elsbach, 1’. (1973) Biochim. Biophys. Acta 296, 365-373
