Induction by lysophospholipids of CoA-dependent arachidonyl transfer between phospholipids in rat platelet homogenates

Biochimica et Biophysics Acta. 793 (1984) 42-48 Elsevier

42

BBA 51600

IN~U~ON BETWEEN ODILE

BY LYSOPHOSPHOLIPIDS OF CoA-DEPENDENT PHOSP~OLIPfDS IN RAT PLATELET HO~O~ENA~S

COLARD,

MICHELYN~

BRETON

and GILBERT

A~CH~DONYL

TRANSFER

BEREZIAT

Laboratoire de Biochimie, Faculti de Mgdecine Sainr - Antoine, 27, rue Chaligny, 75571 Paris C&dex 12 (France) (Received

December

2nd, 1983)

Key words: Arac~~dona~e transfer; Pho.~pho~ipidsyn!hesi~; Lysophosphoi~p~~ Platelet lipid; (RaiJ

Rat platelet homogenates are able to catalyze CoA-mediated, ATP-independent transfer of arachidonic acid from platelet phospholipi~ to added lysophospholipids. Homogenates of platelets prela~Iled with radioactive arachidonic or oleic acid were incubated in the presence of CoA and various lysophosphoiipids. Transfer observed with arachidonic acid-Iabelied platelets was dependent on the i~sophospholipid added. When I-alkenyl- or l-acylly~phospha~dyIeth~olamine was used, there was a more efficient arachidony~ transfer from phosphatidylcholine than from phnsphatidylinosito~ to the phosphatidyiethanolami~e fraction. Lysophosp~tidyl~rine also accepted arachidonyl from phosphatidylcholinc. Addition of lysophosphatidylchoIine resulted in a decrease in the labelling of phosphatidylinositol and to a lesser extent of phosphatidyleth~olamine with concomitant transfer to phosphatidylcholine. Ly~phosphatidylinos~l and Iysophosphati~ acid did not act as substrate for this transfer reaction. Free, non-radioactive arachidonic acid did not compete for the labelled arachidonic acid transfer. This pathway may play a major rofe in the synthesis of arachidonyl species of phosphatidyletha~lamine and phosphatidylserine and for the arachidonyl transfer to the phosp~tidylet~nolamine plasmologen in stim~at~ platelets.

Platelet stimulation by a number of agents induces arachidonie acid release from membrane phospholipids [l-8] and triggers its conversion into biolo~cally active products through the cyclooxygenase pathway (prostaglandins and thromboxanes) [1,9] and the lipoxygenase pathway [l,lO]. Various mechanisms can account for this arachidonate release [ll], including PI breakdown by a PI-specific phospholipase C [12,13] which initiates the PI cycie, and hydrolysis of certain phospholipids by a phosphohpase A, [1,14,15,16]. Abbreviations: PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol~ PE, phosphatidylethanolamine: DPE, diacyIphosphatidylethanolam~n~: PPE, plasmalogen p~osphatidylethanol~~~e; LPA, lysophosphatidic acid; HDL, high-density lipoprotein.

~5-2760/84/$03.~

@ 1984 Elsevier Science Publishers

B.V.

All the platefet phospholipids could be hydrolyzed by this phospholipase A, during platelet activation. In fact, though PC appears to be the main substrate using platelets prelabelled with [*4C]arachidonic acid 14,171,several lysoderivatives have been found in stimulated platelets, such as lyso-PC and lyso-PE [7], fyso-PI [lg] and lyso-PA 1193. In vitro studies have further shown that the specificity of this phospholipase A greatly depended on the physical state of the substrate [20,21]. A~achidoni~ acid is the major fatty acid esterifying the position 2 of PE, PI and PS 122,231. It represents 9.5, 84, 73 and 4758, respectively, of the PPE [24], PI, DPE and PS 1251molecular species in human platelet, but only 25% of the PC molecular species [25]. Despite this arachidonic acid pattern in phospholipids, ~‘4C]arachidonic acid is

43

preferentially esterified into PC and PI from both albumin-bound free fatty acid [3,4,26] and lipoprotein phosphatidylcholine [27,28] compared to the amounts esterified into PPE, DPE and PS. Two mechanisms may be involved in arachidonic acid inco~oration into platelet phospholipids: the Lands pathway [29] and the CoA-mediated arachidonic acid transfer first described by Irvine and Dawson [30] in rat liver microsomes. Lands’ pathway can account for the synthesis of arachidonyl PC, since phospholipase A [14-161, arachidonyl CoA synthetase f31] and acyl-CoA-lyso-PC acyltransferases [32] have been described in platelets. The CoA-mediated transfer is suggested by the arachidonyl transfer from PC to PI observed after phosphatidylcholine exchange between platelets and phosphatidylcholin~-loaded HDL [28,33], by thrombin-induced arachidonyl transfer to PPE 134,173 and by long-term [‘4C]arachidonic acid incorporation [35]. In this work, we investigated the ability of platelet homogenates to transfer fatty acids from their endogenous phospholipids to added lysode~vatives and found that this transfer is very active with respect to arachidonic acid. Materials and Methods The radioactive fatty acids [l-‘4C]oIeic acid (59 Ci/ moi) and [l-‘4C]arachidonic acid were purchased from The Radiochemical Centre, U.K. Phosphatidylethanolamine, Amersham, plasmalogen and phosphatidylinositol were P-L Biochemicals. I-Acyipurchased from lysophosphatidic acid was obtained from Serdary. Phosphatidylserine, l-acyllysophosphatidylethanolamine, 1-acyllysophosphatidylcholine, fatty acid-free albumin, acetylsalicylic acid and bee venom phospholipase A, were obtained from Sigma Chemical Co. Platelet preparation Platelet-rich plasma was prepared by centrifugation at 375 x g for 7 min of fresh titrated whole blood from Wistar rats. Platelets were then isolated by centrifugation at 1400 X g for 15 min, and gently resuspended in tyrode buffer, pH 6.5, without Ca2’ and albumin [36,37]. 0.1 mM acetylsalicyclic acid was added to this platelet

suspension and the preparation was allowed to stand for 30 r&n at room temperature. Platelets were then washed and finally resuspended in the same buffer. The suspension was adjusted to lo9 platelets/ml. ~~eiIing of platelets and preparation of homogenates Washed platelets were incubated with [l“C]arachidonic acid (56 pCi/pmol, 0.5 pCi/ml) in 20 @/pCi ethanol and 0.5% fatty acid-free albumin for 40 min at 37°C in tyrode buffer, pH 6.5. Alternatively, [i4C]oleic acid (59 ~Ci/~mol) was incubated at a concentration of 2 pCi/ml of platelet suspension. After the 40 min labelling period, platelets were washed twice and resuspended in tyrode buffer (2. lo9 platelets/ml). This platelet suspension was then sonicated using an MSE sonifier (setting 1.6 for 2 x 30 s). Lysophospholipid substrates and the transfer reaction l-Acyllyso-PS and l-acyllyso-PI were obtained by hydrolysis of the corresponding phospholipids, PS and PI, using bee venom phospholipase A, [38] as previously described [39]. PE plasmalogen was similarly hydrolyzed by the phospholipase A, to give the l-alkenyllyso-PE derivative. The purity of the plasmalogen products and absence of contamination by the 1-acyl derivative were checked according to Horrocks [40]. After a first migration in chloroform/methanol/water (65 : 25 : 4), the silica gel plates were subjected to concentrated HCl vapours for 10 min and left to evaporate for 1 h before migrating in a second direction with chloroform/methanol/25% NH,OH (60 : 30 : 7). The lysophospholipids were stocked as a chloroform solution or sonicated in water at the concentration of 640 PM. These lysophospholipids, adjusted to the required concentrations, were always sonicated just before use (2 X 30 s set at 1.6). Fatty acyl transfer assay Aliquots of platelet homogenate in tyrode buffer, pH 6.5 (0.25 - lo9 platelets), prelabelled with [l-‘4C]arac~donic acid or [l-*4C]oleic acid, were incubated for 30 min at 37°C in the presence of 50 PM CoA and various amounts of lysophospholipids in a final volume of 0.25 ml.

44

The incubation was stopped by addition of 2 ml chloroform/methanol, 2 : 1. 0.1 M KC1 in 50% methanol was then added and the mixture vigorously shaken. The chloroform layer was evaporated to dryness and the lipid extracts dissolved in chloroform/methanol (1: 1) were applied to TLC plates and developed firstly in chloroform/ methanol/acetic acid/H,0 (75 : 45 : 12 : 6) [41] and secondly in petroleum ether/ethyI ether/ acetic acid (90 : 30 : 1) in order to separate PE thoroughly from the solvent front. Spots, visualized by exposure to iodine vapor or by autoradiography, were scraped from plates into vials; 0.5 ml methanol was added to each vial before scintillation cocktail and the radioactivity was determined in a liquid scintillation counter.

t

Trunsacylation assay

Aliquots of homogenate (corresponding to 0.25 lo9 platelets) were incubated for 30 min at 37% with [l-‘4C]arachidonic acid, 0.1 PCi added in 10 ~1 ethanol and 0.5% fatty acid-free albumin in the presence of 50 PM CoA, 0.5 mM ATP and 32 PM 1-acyl- or l-alkenyllyso-PE. The final volume was 0.25 ml in tyrode buffer, pH 6.5, and the lipids were analyzed as for the fatty acyl transfer assay. Results

After 40 min incubation of rat platelet suspension with [1-t4C]arachidonic acid, the pattern of phospholipid labelling was 45, 34, 19 and 7% for PC, PI, PE and PS, respectively. These results are of the same order as those for suspensions of rabbit platelets [3,16] or human platelets [2,4,33], and human platelet-rich plasma [26,34,42]. The labelling did not change after homogenization procedures in a pH 6.5 buffer, Ca*‘-free. Incubation of [ 14C]arachidonyl labelled rat platelet homogenates for 30 min with 1-acyllyso-PE or l-alkenyllyso-PE and CoA induced a large decrease in labelling of PC and a smaller one in PI labelling conco~tantly with a large increase in the PE fraction (Fig. 1). A similar transfer of arachidonic acid occurred with 1-acyl and with 1-alkenyl, particularly from PC, and this transfer was linear in relation to the amount of lysophospholipid added until the 32 FM saturating concentration. Concerning the distribution of ra-

v----1

20

.

~ 16

32

64 PM

Fig. 1. Transfer of arachidonic acid to PE induced by 1 acyl- or 1 alkenyllyso-PE and CoA in prelabelled rat platelet homogenates. Phospholipids were labelled with [l-‘4C]arachidonic acid as indicated under Material and Methods. After labelhng, the platelets were washed in tyrode buffer, pH 6.5, without Ca2+ and resuspended in the same buffer. Aliquots of homogenate equivalent to 0.25. lo9 platelets (125 ~1) were used per assay and incubated for 30 min at 37°C. 50 ,uM CoA and various concentrations of I-acyllyso-PE (A) or I-alkenyllyso-PE (B) in a final volume of 0.25 ml were added in water. Assays were stopped by 2 ml chloroform/methanol (2: 1). The radioactivity in phospholipid fractions is expressed as the percentage PI; O-0, n, PC; I -V, in these fractions. BPE. Data are the mean& SE. of five separate determinations in A and of two determinations in B. Total radioactivity incorporated into the phospholipids was around 40000 cpm.

dioactivity in the phospholipids, a 9% decrease in PC and an 8% increase in PE were then observed at this saturating lyso-FE concentration. When CoA or lyso-PE were omitted from the incubation medium, changes in radioactivity distribution in PE and PC did not exceed 2%. In order to demonstrate that arachidonyl transfer to lyso-PE acceptors did not occur via the Lands pathway, 10 PM unlabelled arachidonic acid was added in the incubation mixture simultaneously with lyso-PE and CoA; the transfer of

45

TABLE

I

TABLE

II

THE CAPACITY OF PLATELET HOMOGENATES TO ACYLATE l-ACYLOR l-ALKENYLLYSO-PE WITH FREE ARACHIDONIC ACID

ABSENCE OF CoA-MEDIATED PLATELET HOMOGENATES OLEIC ACID

Platelets were isolated and homogenized as described under Materials and Methods. 0.25. lo9 platelets were used per assay containing 0.5% fatty acid-free albumin and 6.8 pM [l14C 1arachidonic acid (0.1 PCi) added in ethanol. The different additions were: 50 PM CoA; 0.5 mM ATP; 32 pM lysophospholipids. Results are given as percentage of the total radioactivity of the starting arachidonic acid.

Phospholipids were labelled with [l-‘4C]oleic acid as indicated under Materials and Methods. After labelling the platelets were washed in tyrode buffer, pH 6.5, and resuspended in the same buffer. Aliquots of homogenate equivalent to 0.25. lo9 platelets (125 ~1) were used per assay and incubated 30 min at 37°C in 0.25 ml final. CoA was 50 PM and lysophospholipids 32 pM. The radioactivity in phospholipid fractions is given as the percentage distribution.

% incorporated into the PE fraction

Additions

Acyllyso-PE + Acyllyso-PE + Alkenyllyso-PE Alkenyllyso-PE

CoA CoA + ATP + CoA + CoA + ATP

% Distribution

0.005 37 0.006 31

Homogenate Homogenate Homogenate

32 l.acyl.LPI

64/1M

16

+ lyso-PE + CoA + lyso-PC + CoA

PE

Ps+PI

PC

14.5 15.5 13.7

16.7 16.4 16.4

68.4 68.1 69.6

of PC labelling was accompanied by a large drop in PI labelling and a smaller drop in that of PE. As was the case with lyso-PE, maximal transfer was obtained with 32 PM of both lyso-PS and lyso-PC, but appears somewhat higher with lyso-PS (lo%, but representing a single experiment) than with lyso-PC (7’%), lyso-DPE (8%) or lyso-PPE (6.5%) (Figs. 1 and 2). In contrast to results with these lysophospholipids, the addition of lyso-PI together with CoA to prelabelled platelet homogenate did not modify the distribution of labelled arachidonic acid in the phospholipids at any of the

arachidonic acid to PE was not inhibited (result not shown). In addition, platelet homogenates were unable to acylate lyso-PE without addition of both CoA and ATP to the incubation mixture (Table I). When ATP was present, 1-alkenyllyso-PE and lacyllyso-PE were reacylated at the same rate. CoA-mediated transfer of arachidonic acid also reacylates 1-acyllyso-PS or 1-acyllyso-PC (Fig. 2). When aracbidonyl-labelled platelet homogenates were incubated with lyso-PS, the increase of radioactivity in PS was concomitant with a decrease thereof in PC. With added lyso-PC, enhancement

16

OLEYL TRANSFER IN PRELABELLED WITH

32 l_acyl_ LPC

64~~

16

32 l_acyl.

64 pM LPS

Fig. 2. The capacity of various lysophospholipids to act as acceptor substrates in the CoA-mediated transfer of arachidonyl moieties: dependence on the concentration of the lysolipids. Platelet homogenate preparations and incubation conditions were as in Fig. 1. Lysophospholipids were prepared as indicated under Material and Methods and sonicated just before use (2 x 20 s at step 1.6). The radioactivity is given as the percentage in the phospholipids. Data are the mean * S.E. of three separate determinations when lyso-PC or lyso-PI were added and one determination in duplicate when lyso-PS was added.

lyso-PI concentrations tested. Lyso-PA was also tested as an acceptor in this transfer reaction. With 32 (LM lyso-PA, no change in the labelling dist~bution was observed. When platelet homogenates were prelabelled with [r4C]oleic acid instead of arachidonic acid, we were unable to demonstrate an oleyl transfer from platelet phospholipids to the lyso-PE or lyso-PC added to the preparation (Table II). Discussion In this report we have shown that rat platelet homogenates were able to catalyze a direct CoAmediated, ATP-independent transfer of arachidonic acid from phospholipids to added lysoderivatives. This transfer was demonstrated by the following facts. When arachidonyl-labelled platelet homogenates were incubated with lyso-PE, lyso-PC or lyso-PS, the dist~bution of the radioactivity shifted from the prelabelled platelet phospholipids to the phospholipids corresponding to the lysoderivatives added (Figs. 1 and 2). This transfer was completed without free aracbidonic acid release, since it was not modified by the addition of non-radioactive arac~donic acid. The transfer did not require ATP, thus excluding the possibility of a combined action with a phospholipase A 2 and an ATP-dependent arachidonylCoA synthesis. The transfer did not occur when platelet homogenates were labelled with [r4CJoleic acid. Consequently, this phenomenon appears to be due to the same specific arachidonyl CoAmediated transfer catalyzed by the acyltransferase operating in reverse as that shown in rat liver microsomes by Irvine and Dawson [30] and in murine thym~ytes by Trotter et al [42]. The fact that the platelet acyltransferase described by McKean et al. [32] reacylated l-acyllysophosphatidylcholine with oleyl-CoA and with arachidonylCoA at the same rate, whereas we found no oleyl transfer, suggests that a particular enzyme may exist for arac~donyl-sp~ific CoA-mediated transfer. Platelets being incubated for 30 min with a 32 PM lysoderivative concentration, we observed an arachidonyl transfer in the same range with lysoPC, lyso-DPE and lyso-PPE and somewhat higher with lyso-PS. With lyso-DPE, lyso-PPE and lyso-

PS, the arachidonyl transferred came essentially from PC and to a less extent from PI to DPE and PPE. In the presence of lyso-PC and in contrast with the results reported by Trotter et al. in thymocytes [42], we found a substantial transfer from PI to PC. It seems that PE also acted as an arachidonyl source for lyso-PC, but this was difficult to prove because of the weak labelling of the PE in relation to the amounts of arachidonyl molecular species in platelet PE. However, given the resulting low specific activity, a small decrease in PE labelling could nevertheless account for a substantial breakdown of PE. Lyso-PS is also a good acceptor for arachidonyl transfer, and a rise of 10% was observed in PS 1abeIling when homogenates were incubated with lyso-PS. It seems that PS cannot serve as a source for arachidonyl transfer. Unexpected results were obtained here with lyso-PI: we were unable to show any shift of arachidonic acid from other phospholipids into PI when lyso-PI was added to the reaction mixture, even though both we ourselves [28] and others [33] observed a transfer of arachidonic acid from PC to PI when whole platelets were incubated with arachidonyl-labelled phosphatidylcholine-loaded HDL. Therefore, in platelets this transfer from PC to PI must occur by the Lands pathway, whereas in rat liver microsomes Irvine and Dawson observed a CoA-mediated arachidonyl transfer from PC to lyso-PI [30]. Also lyso-PA did not act as lyso acceptor in the transfer of arachidonic acid, though lyso-PA could be produced in platelets by a specific phosphatidate phospholipase A z [43]. When labelled arachidonic acid is incubated with washed platelets or platelet-rich plasma, more than 78% of incorporated arachidonate is recovered in PC and PI via the Lands pathway [3,4], whereas arachidonic acid is principally found (70%) in PE and PS [22,23]. This discrepancy may be explained by the fact that PE and PS arachidonyl species are synthesized via the CoA-mediated, ATP-independent transfer pathway. An important point deriving from this discordance between the incorporation and the real content in arachidonic acid is that [‘4C]arachidonic acid-labelled platelets are not available for studying arachidonyl release from PE, since [r4C]arachidonyl PE has a low specific activity and since the arac~donyl transfer

47

from PC and PI would mask the loss of arachidonic acid from PE. The CoA-mediated specific arachidonyl transfer pathway evidenced here in rat platelets could explain the following increase in arachidonyl PPE observed both with stimulated and non-stimulated platelets. (i) Incubation for a long time (19 h) of human platelets without stimulation led to an increase of DPE (11%) and PPE (18%) at the expense of other phospholipids as compared to shorter (15 min or 1 h) incubation times [35]. Under these conditions, the increase in PPE labelling from 1% at 15 min to 11% at 19 h was particularly spectacular. (ii) Rittenhouse-Simmons and co-workers in human platelets [17,44] and Vargaftig et al. in rabbit platelets [45] observed that thrombin or ionophore stimulated arachidonyl transfer from platelet phospholipids to PPE. Such a transfer needs 1ysoPPE. When platelets are stimulated, activation of PPE-specific phospholipase A, before the arachidonyl transfer is suggested by the fact that bromophenacylbromide, an inhibitor of venom phospholipase A,, prevents loss of arachidonate from PC and PI, and its increase in PE [45]. Data on the capacity of platelets to produce 1ysoPPE are contradictory. Using gas-liquid chromatography to study the fatty acid pattern of phospholipids after incubation of human platelets at pH 9.5, Jesse and Cohen 1463were unable to show the presence of fatty aldehydes in the lyso-PE fraction. In contrast, Broeckman et al. [7] found a 14% increase in lyso-PE content, which included 27% fatty aldehydes, in thrombin-stimulated platelets. As 95% of PPE molecules contain arachidonic acid 1241, our results agree with Rittenhouse-Simmons’ hypothesis that the phospholipid precursor for free arachidonic acid required for prostaglandin and thromboxane synthesis in platelets could be PPE [44].

3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Acknowledgements

This work was supported by the Institut National de la Sante et de la Recherche Medicale (CRL 817.006 and PRC 131.002). References 1 Bills, T.K., Smith, J.B. and Biophys. Acta 424, 303-314

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M.J. (1976)

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and

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1,

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39 Colard, 0.. Bard, D., Bertziat, G. and Polonovski, J. (1980) B&him. Biophys. Acta 618, 88-97 40 Horrocks, L.A. (1968) J. Lipid Res. 9, 469-472 41 Tamai, Y., Matsukawa, S. and Satare, M. (1971) Brain Res. 26, 149-154 42 Trotter, J., Flesch, I., Schmidt, B. and Ferber, E. (1982) J. Biol. Chem. 257, 1816-1823 43 Billah, M.M., Lapetina, E.G. and Cuatrecasas, P. (1981) J. Biol. Chem. 256, 5399-5403 44 Rittenhouse-Simmons, S., Russell, F.A. and Deykin, D. (1977) B&him. Biophys. Acta 488, 370-380 45 Vargaftig, B.B., Fouque, F. and Chignard, M. (1980) Thromb. Res. 17, 91-102 46 Jesse, R.L. and Cohen, P. (1976) Biochem. J. 158, 283-287