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Home , Arachidonic acid, Diglyceride, Lysophosphatidic acid

Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3238-3241, July 1979 Biochemistry lipase: A pathway for arachidonate release from human (/) R. L. BELL, DONALD A. KENNERLY, NANCY STANFORD, AND PHILIP W. MAJERUS* Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by P. Roy Vagelos, April 23, 1979

ABSTRACT We provide evidence that the mechanism for demonstrated that this diglyceride of defined com- arachidonate release from stimulated human platelets involves position accumulates within 15 sec of thrombin addition to two : a phosphatidylinositol-specific C (EC 3.1.4.10) and a diglyceride lipase. After incubation of washed human platelets. We have also demonstrated a di- platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl- lipase in the particulate fraction of platelets with 2-arachidonoyl diglyceride per 109 platelets was isolated. Ara- sufficient activity to generate the arachidonate for the "burst" chidonate was released from this substrate by the action of di- of and synthesis that follows glyceride lipase located in the particulate fraction of platelets. thrombin stimulation. We postulate that the unique fatty acid The has a pH optimum of 7.0, is stimulated by composition of PtdIns may explain the fact that only arachi- ions and reduced gutathione, and liberates 31 nmol of fatty acid per min per Ignofplatelet particulate protein. The diglyceride donate is released after thrombin stimulation of platelets; the lipase has sufficient activity to account for the 5-10 nmol of fate of the stearate remains to be elucidated. arachidonate released per 109 platelets upon thrombin stimu- lation. That only arachidonate is released upon thrombin MATERIALS AND METHODS stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 posi- Bovine thrombin was a gift from Craig Jackson. [1-14C]Ara- tion. The lipase activity found in membranes can also chidonate, [1-'4C]oleate, [2-3H], and [9,10-3H(N)]tri- hydrolyze the 1-position fhtty acid. Stearate is not released when olein were purchased from New England Nuclear. Icosate- intact platelets are stimulated with thrombin, and the fate of trayonic acid was a gift from W. E. Scott of Hoffmann-La this fatty acid remains to be elucidated. Roche. Arachidonate and standards were purchased from Several tissues, including blood platelets, exhibit the so-called Nu Chek Prep (Elysian, MN). Diolein, , monolein, egg " effect" when stimulated by appropriate agonists (1). lysolecithin, and egg lecithin were purchased from Sigma. Breakdown of phosphatidylinositol (Ptdlns) plus increased Diazomethane was a gift from Charles Parker. All solvents were incorporation of 32p into PtdIns and are ob- reagent grade or better. served upon stimulation (2, 3). Various theories have attempted Thin-Layer Chromatography (TLC). Prostaglandin products to explain this phenomenon, including that the Ptdlns break- were separated in system C of Nugteren and Hazelhof (11). down serves to produce diglyceride to facilitate exocytosis (1, , , and free fatty acids were sepa- 4). Recently, a specific (EC 3.1.4.10) has been rated in diethyl ether/hexane/ (70:30:1 vol/vol). described in human platelets that cleaves Silica gel plates were purchased from Brinkmann (Polygram from platelet Ptdlns when the cells are stimulated by thrombin. Sil-G). Accumulation of diglyceride was observed within 5 sec of Gas/Liquid Chromatography. Complex were thrombin addition; the diglyceride thus formed disappeared methylated by using BF3 in methanol (Applied Science, State over the next 2 min (4). Previous studies have suggested that College, PA). Free fatty acids were methylated with diazo- arachidonate is made available for prostaglandin synthesis by methane. A 12% diethyleneglycol succinate column on Gas the action of a that cleaves arachidonate from Chrom P (Applied Science) was used in a Hewlett-Packard 402 platelet , primarily (PtdCho) gas chromatograph at 1800C. The fatty acids were identified (5) but also Ptdlns and (PtdEtn) by comparison with known standards. The chromatogram (6-8). Release of radioactive arachidonate from cells previously tracings were photocopied and cut out, and individual fatty acid labeled with [14C]arachidonate was measured in these studies. peaks were weighed and compared to standards to determine However, no phospholipase A2 with sufficient activity to pro- the amount of each fatty acid. vide for the burst of arachidonate metabolism that follows Preparation of Substrates. 2-[1-14C]Arachidonoyl or 2-[1- thrombin stimulation has been demonstrated in platelet ex- 14C]oleoyl PtdCho was prepared biosynthetically from rat tracts. Broekman et al. (9) have measured changes in bulk microsomes by using lysolecithin and 14C-labeled fatty acid phospholipids upon stimulation of platelets with thrombin and essentially as described by Robertson and Lands (12). Labeled found that only PtdIns decreases. Analysis of platelet phos- diglyceride was obtained by treating the with pholipid fatty acid composition has shown a unique composition Bacillus cereus phospholipase C purified as described (13). of the inositides (10). This class of phospholipid is composed Phospholipase C (20 ,g/,umol of substrate lecithin) was incu- primarily of 1-stearoyl-2-arachidonyl Ptdlns. Thus, stimulation bated in 30 mM sodium barbital, pH 7.4, containing 0.12 M of a Ptdlns-specific phospholipase C should result in accumu- NaCl and 1 mM ZnSO4 for 20 min at 370C. Diglyceride was lation of l-stearoyl-2-arachidonyl diglyceride. We have now extracted from residual phospholipid with diethyl ether. Di- The publication costs of this article were defrayed in part by page Abbreviations: PtdIns, phosphatidylinositol; PtdCho, phosphatidyl- charge payment. This article must therefore be hereby marked "ad- ; PtdEtn, phosphatidylethanolamine; TLC, thin-layer chro- vertisement" in accordance with 18U. S. C. §1734 solely to indicate matography. this fact. * To whom reprint requests should be addressed. 3238 Downloaded by guest on October 2, 2021 Biochemistry: Bell et A Proc. Natl. Acad. Sci. USA 76 (1979) 3239 glyceride labeled in the glycerol moiety was prepared as de- 7.! scribed above from lipids extracted from [3H]glycerol-labeled platelets (14). Diglyceride was assayed by using Escherichia coli diglyceride kinase (15). 1,2-Diglyceride was stored in small portions at -20'C in petroleum ether to prevent isomerization to 1,3-diglyceride. That the substrate was maintained as 1,2- i.5.0|. diglyceride was monitored by both TLC and diglyceride ki- nase. on Transient Diglyceride. Human platelets were isolated as described (16). Washed platelets (101") were suspended at 5 X 109/ml in 0.015 M Tris-HCI, pH 7.4, containing 0.14 M NaCl and 5 mM and placed in a separatory funnel containing LL2.5 thrombin (20 units/ml final concentration). After 15 sec, 10 vol of diethyl ether/petroleum ether/acetic acid (80:20:1 vol/vol) was added and the mixture was shaken. The ether layer was removed, 1 nmol of 2-[1-14C]arachidonoyl diglyceride (20,000 0 25 50 75 100 cpm) was added, and the solvent was then evaporated. The Time, min residue was dissolved in chloroform/methanol (9:1 vol/vol) and with the FIG. 1. Time course of diglyceride lipase activity. 2-[1-14C]Oleoyl poured over a 5-g column of silicic acid equilibrated diglyceride (700 AM) was used as substrate with 150 Ag of platelet same solvent. The neutral lipid fraction passed directly through particulate protein as the enzyme source. the column and was collected, the solvent was evaporated, and the lipid was further fractionated by TLC. It was important to minimize the time in chloroform (<30 min) to prevent isom- suggested that it might be the precursor of arachidonate for erization of 1,2-diglyceride to 1,3-diglyceride. The region prostaglandin and thromboxane synthesis. We therefore de- corresponding to 1,2-diglyceride was eluted with 20% methanol termined the level of diglyceride lipase activity in platelets by in diethyl ether and was extracted with aqueous K2CO3 as de- using 2-[1-14C]oleoyl diglyceride or 2-[1-14C]arachidonoyl di- scribed below to remove traces of fatty acid. The diglyceride glyceride. Product was measured by using the two-phase ex- fatty acid was methylated and analyzed by gas/liquid chro- traction method of Belfrage and Vaughn (17); free fatty acid matography. The yield of diglyceride was determined from the is soluble in the basic aqueous phase and diglyceride in the or- recovery of '4C-labeled diglyceride and of C17:0 fatty acid ganic phase. Diglyceride lipase activity was found in platelet standard added prior to methylation. sonicates with >95% of the activity in the 105,000 X g pellet Diglyceride Lipase Assay. Assays were done in 0.1 ml of these sonicates. This particulate preparation was used for containing 50 mM Hepes*NaOH at pH 7.0, 100 mM NaCl, 5 further studies. As shown in Fig. 1, the reaction was linear for mM CaC12, 12 mM reduced glutathione, and either 2-[1- less than 2 min. releasing fatty acid increased with 14C]arachidonoyl diglyceride or 2-[1-14C]oleoyl diglyceride as increasing protein concentration as shown in Fig. 2. A substrate substrate. Assays were started by sonicating enzyme with sub- concentration curve at 2 min is shown as a Lineweaver-Burk strate, using a Biosonic II sonifier with a microprobe, and ter- plot inr Fig. 3. The apparent Km for diglyceride was 0.3 mM, minated by the addition of 1.5 ml of chloroform/methanol/ the maximum velocity of the reaction was 31 nmol of free fatty heptane (1.25:1.4:1.0 vol/vol) and 0.5 ml of K2CO3 (17). Por- acid formed per min per mg of protein. This is sufficient di- tions of the upper aqueous layer were used for scintillation counting in 3A70 fluid (Research Products International, Elk Grove Village, IL). RESULTS Initially we confirmed the finding of Rittenhouse-Simmons (4) that thrombin causes transient accumulation of diglyceride. We postulated that 1-stearoyl-2-arachidonoyl diglyceride would analyzed 75 accumulate, assuming that it arose from PI. When we E the fatty acid composition of transient diglyceride isolated 15 c addition, only arachidonate and stearate -6 sec after thrombin Ca were found. The amount of diglyceride was estimated from the 0) total fatty acids measured by gas/liquid chromatography and a) found to be 1.1 nmol of arachidonate per 109 platelets and 1.3 nmol of stearate per 109 platelets. The isolated diglyceride was shown to be 1,2-diglyceride both by TLC and by the fact that C. it was converted to [32P]phosphatidic acid upon incubation with Al E. coli diglyceride kinase in the presence of [-y-32P]ATP. This experiment was performed three times with platelets pooled from 500 ml of fresh flood from each of two donors. In the second experiment, we recovered 1.2 nmol of arachidonate per 109 platelets and 1.3 nmol of stearate per 109 platelets. In the third experiment no carrier diglyceride was added so that total 200 300 recovery was not calculated. The diglyceride isolated contained Enzyme, ,g 1.2 mol of stearate per mol of arachidonate. FIG. 2. Enzyme concentration curve of diglyceride lipase activity. Diglyceride Lipase. The time course of accumulation and The incubation was for 30 min with various amounts of platelet par- unique fatty acid composition of the transient 1,2-diglyceride ticulate protein and 700 MM 2-[1-14C]oleoyl diglyceride. Downloaded by guest on October 2, 2021 3240 Biochemistry: Bell et al. Proc. Natl. Acad. Sci. USA 76 (1979) 109 platelets) was produced by platelet sonicates, whereas no malonaldehyde was formed when sonicates from - treated platelets were used or when diglyceride was omitted. 0 In order to further characterize the prostaglandin products formed from diglyceride we added 0.1 Mmol of 2-[1-14C]ara- chidonoyl diglyceride to 4 X 109 intact platelets in 1 ml. Ten E minutes after addition of diglyceride and thrombin, 10 units/ ml, we extracted the reaction mixture with acetic acid/ethyl L.50.30- acetate at E pH 3 and separated the arachidonate metabolites by C TLC. We found labeled , 12L-hydroxy- 5,8,10,14-icosatetraenoic acid, and 12L-hydroxy-5,8,10-hep- tadecatrienoic acid by radiochromatography as identified by 0.15- comparison with standard compounds. Thrombin-Induced Release of Platelet Fatty Acids. From the above characteristics of platelet diglyceride lipase, one might expect both arachidonate and stearate to be released from / ~~~I~ ~,I~ 0 15 30 45 platelets after thrombin treatment. Intact platelets (109/ml) 1/diglyceride, mM-' were treated with thrombin (10 units/ml) for 5 min in 5-15 ,uM icosatetraynoic acid to block arachidonate metabolism. The FIG. 3. Lineweaver-Burk plot of substrate concentration curve. fatty acids released were extracted into ethyl acetate at pH 3.0. Assay mixtures contained 25 ,g of platelet particulate protein and In nine experiments we found 6.4 2.4 various concentrations of 2-[1-14C]oleoyl diglyceride. Incubation time ± (SD) nmol of arachi- was 2 min. donate released per 109 platelets; only traces of stearate and other fatty acids were found. Therefore, in intact platelets either stearate is not hydrolyzed or it is sequestered or reutilized im- glyceride lipase activity to account for the 5-10 nmol of ara- mediately. Previous investigations have shown that lysophos- chidonate released per 109 platelets upon thrombin stimulation phatidic acid forms when intact platelets are incubated with (109 platelets yields 1 mg of platelet particulate protein). The thrombin (20). Thus, might be metabolized by enzyme showed similar activity with 2-arachidonoyl or 2-oleoyl a monoglyceride kinase, but because little ATP was present diglyceride. during our diglyceride lipase measurements, this potential re- The enzyme was stimulated by calcium ions as much as action should not occur. 1.5-fold at 5 mM, whereas activity was reduced to 40% by 5 mM EDTA in the absence of added calcium. Because diglyceride DISCUSSION lipase activity was stimulated by reduced glutathione (1.2- to We postulate that prostaglandin and thromboxane synthesis is 3-fold) and reduced to 15% by 0.7 mM N-ethylmaleimide and initiated in platelets by thrombin and other agonists by the to 25% by 0.07 mM p-chloromercuribenzoate, it seems likely pathway shown in Fig. 4. Most workers have assumed that that the enzyme has a sulfhydryl group essential for catalytic arachidonate metabolism is triggered by activation of a phos- activity. The enzyme was active over a broad pH range, pholipase A2. In studies in which [14C]arachidonate was incu- peaking at pH 7.0 and dimishing to less than 20% of maximal bated with platelets to label endogenous phospholipids, it was activity above pH 7.9 or below pH 6.0. concluded that PtdCho and PtdIns were the major phospho- The lipase activity differs from previously described neutral lipids labeled. Upon thrombin stimulation, [14C]arachidonate lipases (18) in that it hydrolyzes poorly, if at all was released primarily from PtdCho. However, these studies (<0.5 nmol of free fatty acid released per min per mg of protein from [3H]triolein). Unlike other lipases, the enzyme appears to cleave fatty acid from the 2 position of diglyceride. The Phosphatidyl Inositol enzyme may also cleave 1-position fatty acid in vitro, because we have been unable to isolate any monoglyceride product. CDP-diglyceride When the enzyme was incubated with diglyceride labeled with 3H in the glycerol backbone and 14C in the 2-position fatty acid, CTP no radioactive monoglyceride was detected. Rather the prod- Phospholipase C ucts were all water soluble, suggesting the formation of [3H]- glycerol and '4C-labeled fatty acid. When the K2CO3 extract inositol phosphatidic ocid containing both the 14C- and 3H-labeled products was acidified ATP /t and extracted with hexane, the 14C-fatty acid was extracted into Diglyceride ? AA-CoA the organic phase but the 3H-product(s) remained in the aqueous phase further, suggesting that free glycerol is produced by the action of this crude diglyceride lipase preparation. [3H]Glycerol and [14C]arachidonate were measured at 5 and 30 min. We found 1 mol of glycerol per mol of arachidonate Diglyceride Lipase at both times. Thus, it appears that stearate and arachidonate are hydrolyzed at the same rate. monoglyceride ATP or glycerol + Malonaldehyde Production from Diglyceride. That the diglyceride lipase pathway can provide arachidonate for prostaglandin synthesis was shown by incubating platelet son- FIG. 4. Proposed pathway for arachidonate release from stimu- icates with 2-arachidonoyl diglyceride and measuring malon- lated platelets. The question marks indicate that the step involving aldehyde as described previously (19). In the presence of 0.6 reincorporation of arachidonic acid (AA) into phosphatidic acid has mM 2-arachidonoyl diglyceride, malonaldehyde (1 nmol per not yet been described in platelets. Downloaded by guest on October 2, 2021 Biochemistry:BBProc.Bell et al. Natl. Acad. Sci. USA 76 (1979) 3241 measured only radioactive arachidonate and the specific ac- amount of diglyceride in these cells and thus ultimately the tivity of the precursor phospholipids was not determined. Re- amount of prostaglandins produced. cently, Broekman et al. (9) showed by quantitative phospho- This research was supported by Grants HLBI 14147 (Specialized lipid analysis that only PtdIns levels decrease after thrombin Center for Research in Thrombosis) and HLBI 16634 from the National treatment. We and others (7, 21) have detected small amounts Institutes of Health and by Medical Scientist Training Program Award of phospholipase A2-like activity in platelet extracts. However, GM 07200 to D.A.K. This work was presented in part at the Deuel the activity detected by using either endogenous substrates or Conference on Lipids, March, 1979, Carmel, CA. added PtdCho or PtdEtn (-0. 1 nmol of fatty acid released per 1. Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147. min per mg of protein) was insufficient to account for the burst 2. Lloyd, J. V., Nishitawa, E. D. & , J. F. (1973) Br. J. of arachidonate required for prostaglandin and thromboxane Haematol. 25, 77-99. synthesis (5-10 nmol of arachidonate per 109 platelets). In ad- 3. Lapetina, E. G., Schmitges, C. J., Chandrabose, K. & Cuatrecasas, dition, we find that the phospholipase A2 activity does not show P. (1977) Biochem. Biophys. Res. Commun. 76, 828-835. the required specificity for 2-arachidonoyl phospholipids, be- 4. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 63,580-587. cause 2-oleoyl phospholipids are hydrolyzed equally well. In 5. Bills, T. K., Smith, J. B. & Silver, M. J. (1977) J. Clin. Invest. 60, contrast, the diglyceride lipase that we describe has sufficient 1-6. to burst of arachidonate released after 6. Schoene, N. W. & Iacono, J. M. (1975) Fed. Proc. Fed. Am. Soc. activity provide for the Exp. Biol. 34,257. platelet stimulation. Arachidonate specificity is not required 7. Blackwell, G. J., Duncombe, W. G., Flower, R. J., Parsons, M. F. for this enzyme because the only unsaturated fatty acid sub- & Vane, J. R. (1977) Br. J. Pharmacol. 59,353-366. strate available in platelets is arachidonoyl diglyceride. Mauco 8. Russell, F. A. & Deykin, D. (1976) Am. J. Hematol. 1, 59-70. et al. (20) recently suggested that platelets contain diglyceride 9. Broekman, M. J., Ward, J. W. & Marcus, A. J. (1979 ) Clin. Res. lipase activity, although no data were reported and the enzyme 27, 459a (abstr.). was postulated to cleave the fatty acid from the 1 position of 10. Marcus, A. J., Ullner, H. L. & Safier, L. B. (1969) J. Lipid Res. diglyceride to yield fatty acid and 2-monoglyceride. 10, 108-114. We assume that thrombin binding to its platelet 11. Nugteren, D. H. & Hazelhof, E. (1973) Biochim. Biophys. Acta results in the activation of a PtdIns-specific phospholipase C. 326,448-461. treatment 12. Robertson, A. F. & Lands, W. E. (1962) Biochemistry 1, 804- Because arachidonate release is inhibited by prior 810. of platelets with agents that elevate cyclic AMP (22), and these 13. Little, C. Aurebekk, B. & Otnaess, A. B. (1975) FEBS Lett. 52, agents also block PtdIns phospholipase C activity (4), it seems 175-179. likely that arachidonate metabolism is triggered by PtdIns 14. Lewis, N. & Majerus, P. W. (1969) J. Clin. Invest. 48, 2114- phospholipase C activation. However, it is also possible that 2123. subsequent steps may also regulate the flow of arachidonate into 15. Kennerly, D. A., Parker, C. W., Sullivan, T. J. (1979) Anal. Bio- prostaglandin and thromboxane formation. For example, chem., in press. competition for diglyceride by lipase and diglyceride kinase 16. Baenziger, N. L. & Majerus, P. W. (1974) Methods Enzymol. 31, may occur and the diglyceride lipase itself may be subject to 149-155. 17. Belfrage, P. & Vaughn, M. (1969) J. Lipid Res. 10, 341-345. modes of regulation yet undiscovered. A number of remaining 18. Brockerhoff, H. & Jensen, R. G. (1974) Lipolytic Enzymes (Ac- questions include: (i) What determines the unique fatty acid ademic, New York), pp. 55-58, 107. composition of platelet PtdIns (ii)? Is arachidonoyl PtdIns 19. Roth, G. J., Stanford, N. & Majerus, P. W. (1975) Proc. Natl. Acad. resynthesized from diglyceride after thrombin stimulation? (iii) Sci. USA 78,3073-3076. What, if any, are the roles of inositol phosphate, monoglyceride, 20. Mauco, G., Chap, H., Simon, M. F. & Douste-Blay, L. (1978) phosphatidic acid, or lysophosphatidic acid in platelet function? Biochimie 60,653-661. (iv) What happens to the stearate portion of the diglyceride? 21. Derkson, A. & Cohen, P. (1975) J. Biol. Chem. 250, 9342- It seems likely that the mechanism in Fig. 4 is a general one. 9347. Hokin and Hokin (23) observed that the pancreas responds to 22. Minkes, M., Stanford, N., Chi, M. M-Y., Roth, G. J., Raz, A., cholinergic agents with an increased turnover in PtdIns. As Needleman, P. & Majerus, P. W. (1977) J. Clin. Invest. 59, is found in nervous 449-454. reviewed recently (1), this phenomenon 23. Hokin, M. R. & Hokin, L. E. (1953) J. Biol. Chem. 203, 967- tissue, pancreas, salivary glands, thyroid, lymphocytes, smooth 977. muscle, and platelets. Tissue mast cells, which function in a 24. Kennerly, D. A., Sullivan, T. J. & Parker, C. W. (1979) J. Im- manner similar to platelets, showed both increased PtdIns munol. 122, 152-159. metabolism (24) and elevated diglyceride levels when stimu- 25. Kennerly, D. A., Parker, C. W. & Sullivan, T. J. (1979) Fed. Proc. lated by agonists (25). Thus, PtdIns turnover could regulate the Fed. Am. Soc. Exp. Biol. 38, 1018. Downloaded by guest on October 2, 2021