Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Biochimica et Biopl~vsica Acta 816 (1985) 265-274 Elsevier

265

BBA 11541

S t u d i e s on the e n z y m a t i c pathways of calcium i o n o p h o r e - i n d u c e d phospholipid degradation and arachidonic acid mobilization in peritoneal m a c r o p h a g e s Adalsteinn Emilsson and Roger Sundler Department of Medical and Physiological Chemistry, University of Lund, P.O. Box 94, S-221 O0 Lund (Sweden) (Received February 21st, 1985)

Key words: Arachidonic acid; Diacylglycerol; Phospholipid metabolism; Ionophore A23187; Ca2+; (Macrophage)

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive CaZ+-depen dent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacyiglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Caz +-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.

Introduction Arachidonic acid is the main precursor of a family of physiologically highly active compounds, which includes prostaglandins, thromboxanes and leukotrienes. The level of unesterified (and therefore available) arachidonic acid is normally kept low both intracellularly and in blood plasma because of efficient esterification into cellular phospholipids. How arachidonic acid is released and to what extent each phospholipid contributes to the release has been the subject of many recent studies (for review see Ref. 1). The results have been Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PAF-acether, platelet activating factor (1-O-alkyl-2-acetylglycerophosphocholine); EGTA, ethylene glycol bis(/3-aminoethyl ether)-N, N, N', N'-tetraacetic acid

divergent, but most investigators believe that arachidonic acid is released by deacylation of diacylglycerols, formed by phosphodiesterase (phospholipase C) attack on PI [2,3] or its phosphorylated forms [3], or by direct deacylation of phospholipids by phospholipase(s) of type A 2 [3-8], although the relative importance of these pathways and the mechanism of activation are still poorly understood. Peritoneal macrophages in primary culture were chosen for the present study. These cells are rich in esterified arachidonic acid and have been shown to release large amounts of cyclooxygenase and lipoxygenase products, including leukotriene C, after exposure to stimuli such as A23187 [9], zymosan particles [10] and antigen-antibody complexes [11]. Since the cellular response" elicited appears to be Ca2+-mediated, it was considered of interest to study the effects of a CaZ+-ionophore to some

0167-4889/85/$03.30 '~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

266 depth. We showed in a previous communication that the Ca2+-ionophore A23187 induced phosphodiesterase attack on polyphosphoinositides accompanied by an increased phosphorylation of PI, besides stimulating phospholipase A activity [3]. In the present study we have directed our attention also to the other phospholipids in attempts to assess their role as donors of arachidonic acid and to identify the pathways involved in the release.

Experimental procedures Radiolabeled chemicals Radioisotopes were supplied by Amersham: [5,6,8,9,11,12,14,15-3H]arachidonic acid (spec. act. 100-135 Ci/mmol), [1-14C]stearic acid (spec. act. 57 mCi/mmol), [1-14C]palmitic acid (spec. act. 57 mCi/mmol), [l(3)-3H]glycerol (spec. act. 2.5 C i / mmol), [Me-3H]choline (spec. act. 77 C i / m m o l ) , [Me-lac]choline (spec. act. 60 m C i / m m o l ) and [1-3H]ethanolamine (spec. act. 9 Ci/mmol). Preparation and labeling and stimulation of macrophages Mouse peritoneal macrophages were prepared by a modification of the procedure described by Cohn and Benson [12]. Outbred female albino mice (Antimex, Stockholm) were used. Resident peritoneal cells were harvested in 4 ml of Medium 199 (Flow Laboratories), containing 1% heat-inactivated fetal calf serum and heparin (20 units/ml), and were plated (approx. 4 . 106 cells/35-mm well) onto plastic 6-well Linbro tissue culture dishes. The cells were incubated in an atmosphere of 5% CO 2 in air. Non-adherent cells were removed 2-3 h after plating and to each dish was then added 1 ml of M 199 containing 10% fetal calf serum. In experiments where cells were labeled with [ 3H]glycerol, [ 3H]- (or [14C]-) choline, or [3H]ethanolamine the radiolabeled substances were added to this medium. Arachidonic acid labeled with 3H was added in M 199 containing 1% serum or 0.68% ( w / v ) bovine serum albumin when 14C-labeled fatty acids were also present. The ~4C-labeled stearic acid and palmitic acid were transferred to the albumin medium from silica particles as described by Spector and Hoak [13]. The latter medium was passed through a

sterile filter (0.22 /~m pore size, Gelman) before use. After labeling for 0.5-24 h, as indicated, the cells were washed several times with phosphatebuffered saline and were then allowed to equilibrate for 0.5-1 h in fresh serum-free M 199 before the start of the experiment. The ionophore A23187 (Boehringer) was added in 5-10 /~1 of dimethyl sulfoxide. To control dishes was added the same amount of dimethyl sulfoxide. In experiments where the concentration of Ca 2 ~ was varied, M 199 (Earle's salt) was replaced by Earle's balanced salt solution. Extraction and analysis Cells were scraped off the dish in l ml of ice-cold 50 mM HC1 and lipids were extracted with 6 vol. of chloroform/methanol (1:1) containing 0.05% 2,6-di-tert-butylp-cresol as an antioxidant. Phase separation was effected by centrifugation after addition of 2 vol. of 50 mM HC1. Lipid standards were added to the extract as carriers and to aid in identification of the lipids. The chloroform layer was withdrawn, taken to dryness under N2, and was then dissolved in 200 ~l of chloroform/methanol (2 : 1, v/v). Phospholipids were separated by thin-layer chromatography on pre-coated plates (Silica Gel 60, Merck). For optimal separation of phospholipids the solvent system c h l o r o f o r m / m e t h a n o l / acetic a c i d / w a t e r (25 : 20 : 3 : 0.3 by vol., solvent system A) was used. This system allowed resolution of all major phospholipids and lysophospholipids and in addition separation of PI from phosphatidylserine. The above system also separated phosphatidic acid from other lipids but in some instances the solvent system c h l o r o f o r m / methanol/acetic acid/water (25 : 10 : 2 : 0.1, by vol.) was used to improve further the resolution of this lipid. When estimates of the plasmalogen content of PE were made, the lipid extract was treated with a HgC12/HC1 reagent as described by Kates [14] and the plasmalogen content of the sample was calculated after thin-layer chromatography. The separation of [3H]- (or [14C]-) choline-labeled lipids was carried out by developing twice in the solvent c h l o r o f o r m / m e t h a n o l / w a t e r (65 : 35 : 6 by vol., solvent system B). PC, sphingomyelin, PAFacether and lysoPC were all well resolved. The separation of lipid standards in solvent systems A

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and B is shown in Fig. 1. Acylglycerols and unesterified fatty acids were analyzed on Silica Gel G thin-layer plates using light petroleum/diethyl ether/acetic acid (50 : 50 : 2, by vol.). Lipids were visualized with 12 vapour and were then scraped into scintillation vials containing 1 ml of m e t h a n o l / H 2 0 (1:1). 10 ml of either Insta-Gel (Packard) or Insta-Gel/toluene (1:1) were then added and the radioactivity was determined by liquid scintillation spectrometry (Model 4530, Packard). The recovery of radioactivity from the thin-layer plates was in the range 80-95%. Water-soluble choline compounds in the polar phase after partitioning were separated by step-

wise elution from small polypropylene columns (Bio-Rad Lab.) containing 1.8 ml of A G 50-X8 resin in H+-form (200-400 mesh, Bio-Rad Lab.). Glycerophosphocholine was eluted with 5 column volumes of 50 mM HC1 and phosphocholine (plus CDPcholine) with a further 10 column volumes of 50 mM HC1. Choline was then eluted with 10 column volumes of 1 M HC1. The corresponding ethanolamine compounds were analyzed by a similar procedure using 3 and 6 column volumes of 50 mM HC1 followed by 6 column volumes of I M HC1. In some experiments phosphoethanolamine and CDPethanolamine were further separated on a column containing 2 ml of AG1-X8 in formate form (200-400 mesh, Bio-Rad Lab.) by elution with a linear gradient of 0-20 mM formic acid [15]. Radioactivity was determined by liquid scintillation spectrometry after addition of 10 ml of Insta-Gel to either 1 ml or 5 ml of sample. Results

Kinetics of arachidonic acid incorporation and the pattern of A23187-induced mobilization To examine the labeling of individual phospholipid classes, macrophages were exposed to tritiated arachidonic acid for various time periods up to 24 h. As shown in Fig. 2, most of the radioactivity was initially recovered in PI and PC but increased gradually in PE and phosphatidylserine. Noteworthy is the slow but steady increase in the labeling of the PE plasmalogen. In several separate experiments we found the plasmalogen to constitute 50-70% of the total ethanolamine phosphc~lipid. Labeling with [3H] ethanolamine gave siniilar results (not shown). A high content of the plasmalogen form of PE has previously been found in rabbit alveolar and guinea pig peritoneal macrophages [16,17]. Also when radiolabeled stearic and palmitic acid were used a larger fraction of the incorporated radioactivity was recovered in PE after 24 h as compared to 2 h of labeling (see Fig. 4A-C). Since the total incorporated activity reached a maximum after 8 h of labeling and then declined (Fig. 2), the continued increase in phosphatidylserine and PE indicates that there was a slow transfer of activity from PC and PI. To explore this further we prelabeled cells with [3H]arachidonic acid for 2 h and looked at i

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Fig. 2. Time-course of arachidonic acid incorporation into macrophage phospholipids. Macrophages were exposed to 1 FCi of [3H]arachidonic acid for 0.5 24 h and were then harvested, extracted and analyzed for lipid radioactivity. All cultures were terminated at the same time. Symbols: PC (A), PI (O), PE (diacyl plus alkyl-acyl) (O), PE (alkenyl-acyl) (~) and phosphatidylserine (I).

the fate of incorporated activity during further culture in non-labeled medium. As shown in Fig. 3 proportionally more activity was found in PE and its plasmalogen form after 24 than after 2 h, or about twice as much. At the same time a larger fraction of the total activity released with 2 FM ionophore A23187 stemmed from the ethanolamine-containing phospholipids.

Time course of ionophore-induced phospholipid breakdown and arachidonic acid release Diacylglycerol and monoacylglycerol have been considered to be intermediates in arachidonic acid release from phosphoinositides. An increased labeling of these iipids was consistently observed when arachidonic acid-labeled macrophages were treated with ionophore A23187. In order to identify the source(s) of these intermediates in a more

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Fig. 3. Redistribution of [3H]arachidonic acid between phospholipids: effect on ionophore stimulation. Peritoneal macrophages were labeled with 1 p.Ci [3H]arachidonic acid for 2 h and were then cultured for various times after removal of unincorporated isotope. All cells were harvested at the same time. A parallel set of cell cultures were treated the same way, except that ionophore A23187 was added 15 min before harvesting. A, PC ( O , O ) and PI (zx, A); B, PE ( O , o ) and phosphatidylserine (zx, A). lonophore-treated cells are indicated by filled symbols and non-stimulated control cells by open symbols.

direct way we carried out experiments where macrophages were labeled with both [3H]arachidonic acid and [14C]stearic acid for 2 or 24 h. We then compared the results from these cultures with those from parallel cultures where the cells were labeled with [14C]palmitic acid. The time course of fatty acid release induced by 2 ~m A23187 is shown in Fig. 4. After 2 h of labeling arachidonic acid was rapidly liberated from PC and PI with maximal release within 15 min. At that time PC had lost about 45% of its incorporated activity and PI about 40%. PE lost its activity more slowly and leveled off at a decrease of 40% of its original activity. All the major phospholipids lost a fraction of their stearic acid content, whereas palmitic acid was released almost solely from PC. It is noteworthy that arachidonic acid and stearic acid were apparently released from PI in parallel, with a concomitant accumulation in diacylglycerol (Fig. 5). This is consistent with a removal of intact diacylglycerol units by phosphodiesterase attack on PI or its phosphorylated forms. The [~4C]-

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Fig. 4. Time-course of ionopbore-induced fatty acid release. Macrophages were prelabeled simultaneously with 5 /~Ci [3H]arachidonic acid and with 1 vCi [14C]stearic acid, or in separate cultures, with 2.5/~Ci [n4C]palmitic acid for either 2 or 24 h. The cells were then stimulated with 2 vM A23187 for the time indicated. A - C , PC; D - F , PI and G - l , ethanolamine phospholipids. O, cultures labeled for 2 h; A, cultures labeled for 24 h; m, alkenyl-acyl form of ethanolamine phospholipids labeled for 24 h. The cells incorporated approx. 15-24% of added [3H]arachidonic acid and 4-17% of added [14C]stearic and palmitic acids.

pholipid became more pronounced (Fig. 4). The radiolabeled arachidonic acid released from ethanolamine phospholipids was about equally contributed by the diacyl- and alkenyl-acyl forms. At the same time the release from PC became less prominent and the release from PI was strongly reduced. This is in line with the disappearance, after 24 h of labeling, of the initial rapid rise in diacylglycerol radioactivity seen after 2 h of labeling (Fig. 5A, B).

palmitic acid-labeled diacylglycerol most likely has another origin, as the rise occurred later, and since this fatty acid was almost absent from PI. The diacylglycerol formed by stimulated phospholipid degradation may be partly deacylated to produce monoacylglycerol. The greater rise in arachidonic acid labeling of the monoacylglycerol compared to the labeling by the saturated fatty acids indicates that the initial attack occurred on the sn-1 position of the glycerol backbone, with retention of the unsaturated acid (Fig. 5). The release of [3H]arachidonic acid from the major phospholipids was altered after 24 h of prelabeling. Concomitant with the increase in arachidonic acid labeling of PE (and its plasmalogen form) the liberation from this phos-

Metabolism of the glycerol backbone and formation of phosphatidic acid An increased labeling of phosphatidic acid after stimulation with ionophore A23187 has been observed in experiments employing several different radiolabeled precursors. Most of this phosphatidic acid was probably formed by phosphorylation of diacylglycerol units released from phospholipids. However, other experiments have shown that the activity in the phosphatidic acid fraction increases also when the ionophore is added during the initial incorporation of radiolabeled fatty acids (not shown), as has been found during glycerol labeling in guinea pig neutrophils [8]. We interpret this as being due to partial inhibition of the further metabolism of phosphatidic acid, rather than to enhancement of de novo synthesis [8]. In pre-

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phages prelabeled with tritiated inositol by analyzing the water-soluble degradation produces formed upon stimulation [3]. In attempts to use a similar experimental approach also for the other phospholipids from which a loss of arachidonic acid occurred, macrophages were prelabeled with either [ 3 H ] - (or [14C]-) choline or [3H]ethanolamine before exposure to 10 /~M A23187 for 5 rain. The results from such experiments are summarized in Table I. About 10% of the lipid-bound choline was converted into glycerophosphocholine, phosphocholine and choline, the latter most probably generated by further degradation of the former compounds. A somewhat larger fraction of the labeled ethanolamine-containing lipids was broken down under the same conditions but in this case only phosphoethanolamine and CDPethanolamine were detected. Separation of these two compounds showed that the latter accounted for 1% of the total radioisotope content and increased in proportion to phosphoethanolamine (not shown). The time course of degradation of cholinelabeled PC and the appearance of water-soluble as

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Fig. 6. Ionophore effects on the metabolism of [3H]glycerollabeled macrophage phospholipids. Macrophages were prelabeled with 125/~Ci of [3H]glycerol for 24 h. After change of medium the cells were stimulated with ionophore A23187 (10 #M). A, diacylglycerol (O), monoacylglycerol (I), and phosphatidic acid (A); B, PC (o), PI (A) and PE (I). The total recovered activity was 1.8.106 dpm per dish. Transesterification in methanol/BF3 indicated that less than 1% of the radioactivity in the lipid extract resided in the fatty acids.

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271 TABLE I IONOPHORE A23187-1NDUCED DEGRADATION OF CHOLINE- AND ETHANOLAMINE-CONTAINING PHOSPHOLIPIDS AND THE ACCUMULATION OF DEGRADATION PRODUCTS Macrophages were prelabeled with 5 #Ci [3H]choline or 2 /~Ci []4Clcholine or with 10 #Ci [3 H]ethanolamine for 24 h. The cells were then stimulated with 10 /~M A23187 for 5 min. Values shown represent percent of total recovered radioactivity for control cells ( i S . E ) and changes from these values upon stimulation with the ionophore (±S.E.). Values for phosphocholine and phosphoethanolamine include CDPcholine and CDPethanolamine, respectively.

Total lipids Glycerophosphocholine (-ethanolamine) Phosphocholine (-ethanolamine) Choline (ethanolamine)

[ 3H]Choline/[14 C]choline

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A23187 (n = 6) (change from control)

control (n = 3) (% of total)

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a 100% activity denotes 1.2.10 6 (n = 3) and 3.3.105 dpm (n = 6) per dish for 3H- and 14C-labeled cultures, respectively. b 100% activity denotes 4.10 6 dpm per dish. P < 0.05 determined by Student't t-test for paired samples. d p < 0.01 determined by Student's t-test for paired samples. well as l i p i d - s o l u b l e p r o d u c t s is s h o w n in Fig. 7. T h e d i s a p p e a r a n c e o f [ 3 H ] c h o l i n e f r o m P C foll o w e d a t i m e c o u r s e s i m i l a r to t h a t for a r a c h i d o n i c a c i d in the e x p e r i m e n t s d e s c r i b e d a b o v e , w i t h a b o u t 20% of the i s o t o p e b e i n g r e l e a s e d d u r i n g 15 m i n of stimulation. The deacylation product glycerophosp h o c h o l i n e was r e l e a s e d r a p i d l y , p r o b a b l y b e c a u s e of e f f i c i e n t d e g r a d a t i o n o f l y s o P C , w h i c h a c c u m u l a t e d o n l y s l o w l y d u r i n g the o b s e r v a t i o n p e r i o d . T h e p h o s p h o c h o l i n e r a d i o a c t i v i t y was i n i t i a l l y unc h a n g e d b u t s t a r t e d to rise a f t e r 5 rain o f s t i m u l a tion. S u r p r i s i n g l y w e o b s e r v e d a r a t h e r s h a r p 25% i n c r e a s e in the s p h i n g o m y e l i n f r a c t i o n t h a t we i n t e r p r e t as an i n c r e a s e d t r a n s f e r of p h o s p h o c h o line units d e r i v e d f r o m P C [18]. P A F - a c e t h e r has b e e n s h o w n b y o t h e r s to b e g e n e r a t e d u p o n s t i m u l a t i o n o f the m a c r o p h a g e s by the i o n o p h o r e u s e d h e r e [19]. H o w e v e r , u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s P A F - a c e t h e r was o n l y a q u a n t i t a t i v e l y m i n o r p r o d u c t of c h o l i n e p h o s p h o l i p i d b r e a k d o w n .

The role o f extracellular Ca z + in ionophore-induced arachidonic acid release and phospholipid breakdown In o r d e r to a s c e r t a i n t h a t the A 2 3 1 8 7 - i n d u c e d

p h o s p h o l i p i d d e g r a d a t i o n was d u e to its C a t+ionophoretic property, experiments were carried o u t at v a r y i n g c o n c e n t r a t i o n s of e x t r a c e l l u l a r C a 2+

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in the absence and presence of the ionophore. Figs. 8 and 9 show the results from such experiments using cells labeled with [3 H]arachidonic acid and [14C]choline. It is evident that the breakdown of arachidonic acid-labeled PC and PI and the appearance of lipid soluble products were absolutely dependent on Ca 2+, with practically no degradation in Ca2+-free medium containing E G T A (Fig. 8). The release of arachidonic acid was stimulated even at 50 ~m extracellular Ca 2~ and reached a maximum at 1 m M Ca 2+ concentration. The breakdown of [14C]choline-labeled PC and the appearance of deacylation products was not detectable at low Ca 2+ concentrations, but at 0.5 m M extracellular Ca 2+ significant deacylation occurred (Fig. 9). Most likely, the reacylation of lysoPC prevented the accumulation of deacylation products at lower rates of arachidonic acid release. Free choline showed behaviour similar to that of the deacylation products at low Ca 2+ concentrations, but continued to rise up to 1.8 m M Ca 2+.

Resident peritoneal macrophages respond promptly to several soluble and particulate stimuli by producing oxygenated arachidonic acid metabolites [10,11,20] and secreting hydrolytic enzymes. Furthermore, the macrophages can be held in culture for relatively long periods of time. The latter is an advantage in studies of arachidonic acid mobilization since, as shown here. many hours of labeling, or of continued culture after the labeling period, are needed for homogeneous labeling of the various arachidonate-containing phospholipids. Short-term radioisotope labeling may therefore give rise to misleading results with regard to the phosholipid source of mobilized arachidonic acid. In particular, the quantitative significance of PI as a source of arachidonic acid can be overestimated after short-term prelabeling. Pl incorporates arachidonic acid rapidly and reaches its highest specific activity between 4 and 8 h of labeling (see also Ref. 21). Studies on thrombocytes [22] indicate that in these cells homogeneous labeling of arachidonic acid pools is not obtained either after short-term labeling. Our results show that arachidonic acid is mobilized not only from PI and PC but also from PE in response to A23187. The mobilization is strictly dependent on extracellular Ca 2+ and can therefore be ascribed, directly or indirectly, to the ionophore-induced increase in intracellular Ca 2' concentration. Several different radiolabeled precursors were used in attempts to gain information about the enzymatic pathway(s) involved in the Ca 2 t-induced phospholipid degradation. Diacylglycerol, generated from inositol phospholipids, has been considered a central intermediate in the process of arachidonic acid release in thrombocytes [2]. However, in our system the diacylglycerol which accumulated in response to the Ca2+-ionophore had more than one origin. Comparative experiments with radiolabeled fatty acids showed that the rapid initial rise in diacylglycerol in cells prelabeled for 2 h with [3H]arachidonic and [14C]stearic acid correlated well to a corresponding loss from PI, while the somewhat slower labeling observed also with [14C]palmitic acid must have originated in PC and PE. As a corollary to the latter, a considerable part of the radiolabeled

273

choline and ethanolamine released was recovered as phosphocholine and phosphoethanolamine, respectively. Indeed, phosphoethanolamine and CDPethanolamine were the only water-soluble ethanolamine compounds which accumulated. We believe that these products and the corresponding choline compounds were generated by the reversal of the reactions catalyzed by ethanolamine- and cholinephosphotransferase, respectively. The latter reaction has previously been shown to be freely reversible in liver cells [23]. An additional process which became activated and which would generate diacylglycerol from PC is the reaction in which phosphocholine is transferred from PC to ceramide to form sphingomyelin [18]. This reaction was here manifested as a stimulus-induced increase in sphingomyelin labeling in cells prelabeled with [3H]choline. The release of arachidonic acid from diacylglycerol may require an initial removal of the saturated fatty acid at the sn-1 position of the glycerol moiety [24,25], since only a slight increase in stearic and palmitic acid labeling of monoacylglycerol was observed, compared to the large increase in labeling by arachidonic acid. The diacylglycerol formed may also become phosphorylated and an increased labeling of phosphatidic acid was observed early in the time course. The phosphorylation appeared to be most effective at low extracellular concentrations of Ca 2 +, since diacylglycerol accumulation was prevented under these conditions. Direct evidence for a phospholipase A-catalyzed deacylation of PC was also obtained in the present study. The deacylation process was absolutely dependent on extracellular Ca 2+, as there was no increased formation of lysoPC or glycerophosphocholine in the absence of Ca 2+ but a gradual increase in their formation was seen up to 0.5 mM concentration. We have previously shown that the deacylation of PI is enhanced upon stimulation with A23187 [3]. Whether the same phospholipase A is involved in the degradation of PC and PI is not clear at present. In contrast, we have been unable to detect any increased formation of glycerophosphoethanolamine. A comparison of the fate of the choline and arachidonic acid labels in cells prelabeled for 24 h shows that the ionophore-induced loss of both

isotopes from PC is of the same magnitude; i.e., 21-25% during 15 min. This indicates that the degradation of PC via all three of the enzymatic pathways described above contributes to the release of arachidonic acid in ionophore-treated cells. Previous studies on phospholipid degradation and arachidonic acid mobilization induced by ionophore A23187 in cells such as human neutrophils [26,27] and guinea pig peritoneal macrophages [28] and neutrophils [8] have in part succeeded in identifying the enzymatic pathways activated, since an increased formation of diacylglycerol and phosphatidic acid was observed in macrophages [28] and neutrophils [8] by the use of radiolabeled glycerol and arachidonic acid. In the latter system increased formation of 22P-labeled lysoPI and tysoPC, but hardly at all of lysoPE, was also observed, in agreement with the present findings. By analyzing also the polar degradation products of the phospholipids we have been able to show that diacylglycerol becomes generated by more than one enzymatic process and to provide direct evidence for a quantitatively significant complete deacylation of PI [3] and PC (present paper).

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (03X-5410 and 03P-6848), the A. Phhlsson and the A. Osterlund Foundations and the Medical Faculty, University of Lund. Excellent technical assistance by Jonny Wijkander and secretarial assistance by Gesa Johnson are gratefully acknowledged.

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