Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2955-2958, June 1978

Medical Sciences

Low concentrations of indomethacin inhibit phospholipase A2 of rabbit polymorphonuclear leukocytes (inflammation/prostaglandin synthesis)

LESLEY KAPLAN, JERROLD WEISS, AND PETER ELSBACH* Department of Medicine, New York University School of Medicine, New York, New York 10016

Communicated by Michael Heidelberger, March 30,1978

ABSTRACT Inhibition of grostaglandin synthesis b indomethacin, a drug with anti-infammatory properties, has bn attributed to its action on fatty acid cyclooxygenase. However, pwstaglandin synthesis would also be inhibited if precursor fatty acids were not supplied. We find that indomethacin inhibits phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of rabbit polymorphonuclear leukocytes in dose-dependent fashion. Inhibition is immediate and readily detected at 1 ,sM. The extent of inhibition is the same over a 10-fold range of substrate concentration and over a 500-fold range of enzyme purification. Inhibition is of the noncompetitive type, with an apparent Ki of 12 ;M. Four other phospholipases A2-from venoms of Russell viper, Crotalus adamanteus, and bee, and from pig pancreas-are unaffected by 50 ;M indomethacin, which inhibits leukocyte phospholipase A2 by 70%. This inhibition at low concentrations may well be important in the effects of the drug on prostaglandin synthesis and inflammatory responses. Anti-inflammatory agents such as indomethacin and aspirin inhibit prostaglandin synthesis by all cells and tissues so far examined (1-3). This has been attributed to a direct effect on the cyclooxygenase that converts arachidonic acid to prostaglandins (4, 5). However, indomethacin and related compounds that are known to inhibit numerous enzymes (6) may also act on other steps in the synthesis of prostaglandins. Because the regulation of prostaglandins under physiological conditions must include the repletion of the exceedingly small intracellular pool of unesterified arachidonic acid by the action of lipolytic enzymes, we entertained the possibility that indomethacin might also act on the synthesis of prostaglandins by an effect on phospholipases of inflammatory cells. We report that indomethacin at 1 MM inhibits phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of rabbit polymorphonuclear leukocytes (PMN) with apparent specificity, because four phospholipases A2 from other sources are unaffected by indomethacin at 50 AM.

MATERIALS AND METHODS Collection of PMN. PMN were obtained from sterile rabbit peritoneal exudates prepared by injection of 0.1% glycogen in saline as previously described (7). Preparation of Fractions I-IV with Phospholipase A2 Activities of Different Purity. Procedures a-c have been recently described (8). Procedure d is to be published in detail. (a) Homogenization. Fraction I is prepared from freshly collected PMN that are washed, resuspended in distilled water to a concentration of 3 X 108 cells per ml, and homogenized as described before (8). (b) Acid extraction. Fraction II is obtained by treatment of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

whole homogenate with 0.08 M H2SO4. After neutralization of the extract by dialysis against 1 mM Tris-HCl buffer (pH 7.4) and removal of an inactive precipitate by centrifugation at 23,000 X g for 20 min, the clear supernatant contains from 80 to 120% of the original phospholipase A2 activity (8). (c) Chromatography on carboxymethyl-Sephadex. This procedure (8) produces a discrete protein fraction (fraction III) with all of the recovered phospholipase A2 activity (40-

50%). (d) Gel filtration. Chromatography on Sephadex G-50 of fraction III separates a high molecular weight component devoid of phospholipase A2 activity from a lower molecular weight mixture (molecular weight < 25,000) with all phospholipase A2 activity of the eluate (fraction IVa), but concentrated in a small subfraction (fraction IVb). Preparation of Radioactive Substrates. The phospholipids of Escherichia coli strain S15 were labeled during growth with [1-14C]oleic acid (specific activity 40 Ci/mol, Amersham/Searle Company) as recently described (9). After labeling, the organisms were autoclaved for 15 min at 120° and 2.7 kg/cm2 (260 kPa). This inactivates heat-stable bacterial phospholipases (10) and renders the phospholipids of the envelope readily accessible to the action of added phospholipases A. Autoclaving does not detectably alter the thin-layer chromatographic properties of the phospholipids or the composition of their fatty acids as determined by gas-liquid chromatography (11). More than 90% of the incorporated oleic acid occupies the 2 position of E. coli's main phospholipids, phosphatidylethanolamine and phosphatidylglycerol, as determined by subsequent degradation with boiled Russell viper venom phospholipase A2 (11), so that the action of phospholipase A2 can be monitored by measuring release of free [14C]oleic acid. Assay of Phospholipase A Activity. Assay mixtures of 0.5 ml contained 80 mM Tris-HCl buffer (pH 7.5), 10 mM CaCl2, 20 Ag of PMN fraction II (unless specified otherwise), and the indicated amounts of phospholipid substrate. Incubation was carried out at 370 for periods up to 15 min and terminated by addition of 3 ml of methanol/chloroform (2:1; vol/vol). Extraction of lipids (12), their fractionation by thin-layer chromatography in ether/petroleum ether/glacial acetic acid (80:20:1; vol/vol), and assay of radioactively labeled fractions were carried out as in ref. 10. Hydrolysis of phospholipids is expressed as % of total lipid radioactivity recovered in the free fatty acid fraction. Protein was determined as in ref. 13.

RESULTS Fig. 1 shows that indomethacin inhibits the hydrolysis of E. coli phospholipid by partially purified phospholipase A2 from rabbit PMN (fraction II) (8-10). The rate of release of oleic acid in the Abbreviation: PMN, polymorphonuclear leukocytes. * To whom reprint requests and inquiries should be addressed.

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Proc. Natl. Acad. Sci. USA 75 (1978)

I.

.Co z 1.0

o 0.4 E 0.2

c

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FIG. 1. Inhibition of phospholipid hydrolysis by indomethacin. Reaction mixtures of 0.5 ml contained 2.5 X 108 autoclaved [1 -4C]oleic acid-labeled E. coli, representing 5 nmol of phospholipid, and 4 Al (20 ,ug of protein) of fraction II, which contains an amount of enzyme that produces approximately 20% hydrolysis of the added substrate in 15 min. Indomethacin freshly dissolved in absolute alcohol was added in a final concentration of 10 (A) or 25 (o) MM. Control( ) ,no indomethacin. Alcohol alone in equivalent amounts is without effectonphospholipid hydrolysis. The results represent the means of three closely similar experiments.

absence of indomethacin falls off after the first S min of incubation, but the inhibitory effect of indomethacin, added at zero time, on hydrolysis is as pronounced at S min as at 15 min. That the action of indomethacin on hydrolysis is immediate is further indicated by the fact that inhibition is not enhanced by preincubation of the enzyme with indomethacin for 15 min. Inhibition of hydrolysis by indomethacin is dose dependent: at 10 WM indomethacin it was approximately 40%, and at 25 WM, 60% (Fig. 1). Hydrolysis is abolished almost entirely by 100MIM indomethacin. The lowest indomethacin concentration that reproducibly inhibits hydrolysis [by 14 1 1% (mean : SEM) in four separate experiments] is 1 MAM. The extent of inhibition of hydrolysis by indomethacin is not dependent on the concentration of substrate over an approximately 10-fold range (Fig. 2), indicating that the drug interacts with the enzyme rather than the substrate. In Fig. 3 release of fatty acid from increasing amounts of substrate during 5-min incubation [initial rapid rate (Fig. 1)] with fraction H alone and with 10 and 25 MAM indomethacin is plotted according to Lineweaver and Burk (see ref. 5), yielding an apparent Km of 7MuM and an apparent Ki of 12MAM. Inhibition by indomethacin appears to be of the noncompetitive type.

The inhibitory effect of the drug, measured at concentrations from I to 400,uM, is not appreciably modified by purification of the PMN phospholipase A2 to approximately 500-fold increase in specific enzymatic activity (Fig. 4 and Table 1). In contrast to the inhibition of PMN phospholipase A2 by concentrations of indomethacin as low as 1 MAM, hydrolysis of E. coil phospholipids by four phospholipases A2 from other sources is unaffected by indomethacin at 50MgM. Inhibition is observed, however, of Russell viper venom and bee venom phospholipase at a concentration of 100 MmM, and all of the phospholipases A2 are profoundly inhibited by 1 mM indomethacin (Table 2).

DISCUSSION Indomethacin inhibits numerous enzymatic activities in vitro (6). Many of these effects are evident only at relatively high concentrations and their pharmacological significance is uncertain. The inhibitory effect of indomethacin on prostaglandin synthesis by many cells, including PMN, however, is recognized 100k cl

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10-5 lo-4 10-3 [Indomethacin], M FIG. 4. Effect of purification of PMN phospholipase A2 on its inhibition by indomethacin. Inhibition of phospholipase A2 activity of increasing specific activity (Table 1), by various concentrations of indomethacin (1-400.uM), was determined as already described. The amount of each phospholipase preparation used in the assay was chosen to yield approximately 20% hydrolysis in 15 min, using 2.5 X 108 autoclaved E. coli (5 nmol of phospholipid). Inhibition is expressed as % of hydrolysis in the absence of indomethacin. o, Fraction I; A, fraction II; A, fraction III; 0, fraction IVa; 0, fraction IVb. 10 6

pholipid hydrolysis by indomethacin. Experiments were carried out described in Materials and Methods and in the legend of Fig. 1, except that substrate concentrations varied from 1.5 X 108 to 2 X 109 autoclaved E. coli, representing from 3 to 40 nmol of phospholipid. Each point represents the mean of at least three separate determinations. 0, Hydrolysis in the absence of indomethacin; a, plus 10 AM indomethacin; a, 25 MAM indomethacin; A, 50MgM indomethacin.

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4 8121620 40 nmol phospholipids FIG. 2. Effect of substrate concentration on inhibition of phos-

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FIG. 3. Noncompetitive inhibition of PMN phospholipase A2 by indomethacin. Incubations were carried out as described in the legends of Figs. 1 and 2 except that the substrate concentration was varied as indicated and that the reaction was terminated after 5 min. 1/V = 1/nmol of [1-14C]oleic acid released. 1/S = 1/nmol of substrate (as autoclaved E. coli, 2.5 X 108 to 2 X 109 organisms, or 5 to 40 nmol of phospholipid). 0, Hydrolysis in the absence of indomethacin; a, plus 10 AM indomethacin; E, 25 MM indomethacin. The data were obtained in at least three separate experiments. The lines were drawn on the basis of regression analysis.

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Proc. Nat!. Acad. Sci. USA 75 (1978)

Table 1. Specific activity of phospholipase A2 in various PMN fractions

Specific activity, units/mg protein 2,500 20,000 125,000 50,000 1,250,000 Phospholipase activity, measured against 2.5 X 108 autoclaved [1-14C]oleic acid-labeled E. coli, is expressed in arbitary units [% hydrolysis of 5 nmol of E. coli phospholipid in 60 min (8)]. Fraction I II III IVa IVb

at concentrations in vitro as low as 1 uM, (5, 6) and may be related to its anti-inflammatory action in vwo (5). Thus, prostaglandin synthesis by PMN is stimulated manyfold during phagocytosis and this stimulation is abolished by indomethacin (2). Evidence has been presented indicating that indomethacin interferes directly with endoperoxide formation from arachidonic acid by interacting with cyclooxygenase (4, 5). Because in all tissues examined the intracellular free fatty acid pool under physiological conditions is vanishingly small, precursor polyunsaturated free fatty acids are thought to stem from complex cellular lipids, hydrolyzed when acylhydrolases are activated by the proper stimulus (5). Therefore, depletion of the precursor pool by inhibition of acylhydrolase activity would also cause reduction in prostaglandin synthesis (5, 14). Our results show that indomethacin, at the lowest concentrations inhibitory to prostaglandin synthesis, also inhibits a potent leukocyte phospholipase A2. In the light of these observations it is now uncertain whether in PMN, and quite possibly in other cells, inhibition of prostaglandin synthesis is a consequence of an effect of the drug on the endoperoxide synthetase system alone or the result of inhibition of phospholipase A2, which would limit the availability of precursor polyunsaturated fatty acids. It must be stressed that not all the conversion of arachidonic acid to oxygenated products is inhibited by indomethacin. For example, in intact leukocytes indomethacin has no inhibitory effect on the conversion of arachidonic acid to OH-fatty acids by lipoxygenase (15). This need not imply, however, that inhibition by indomethacin of phospholipase A2 Table 2. Inhibition by indomethacin of phospholipid hydrolysis by various phospholipases A2

Source of

phospholipase A2 Russell viper venom Bee venom Pig pancreas Crotalus adamanteus venom PMN phospholipase A2

% inhibition with final indomethacin concentration of

50 AM 100MM 0 0 0 0 70

37 33 0 0 100

1000,4M 100 100 63 43

Incubations were carried out for 15 min as described in Materials and Methods. Of each phospholipase preparation an amount was added to the incubation mixture sufficient to produce approximately 20% hydrolysis of 5 nmol of [1-14C]oleic acid-labeled E. coli phospholipid in 15 min, in the absence of indomethacin. Venoms from Russell viper (Vipera russelli) and eastern diamondback rattlesnake (Crotalus adamanteus) were obtained from Sigma Chemical Co. Purified bee venom phospholipase A2, freed of melittin, was a gift of R. Zwaal, Department of Biochemistry, University of Utrecht, and purified pig pancreas phospholipase A2 was a gift of C. Dutilh and G. de Haas, Department of Biochemistry, University of Utrecht.

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is unrelated to inhibition by this agent of endoperoxide synthesis. Arachidonate substrate for lipoxygenase and cyclooxygenase may derive from different pools of esterified arachidonate, hydrolyzed by different acylhydrolases with differing susceptibility to indomethacin. For example, approximately 20% of leukocyte lipids are triacylglycerols (7), and during incubation of PMN with [1-14C]arachidonic acid for 1 hr about 20% of esterified 14C-labeled fatty acid appears in this lipid fraction (unpublished observations). Hydrolysis of this repository of polyunsaturated fatty acids in intact PMN has been demonstrated (16). It remains to be determined whether or not the potent leukocyte lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) (17) presumably responsible for this hydrolysis is sensitive to low concentrations of indomethacin. It is also possible that conditions exist, especially in vitro, permitting extracellular unesterified arachidonic acid to reach the lipoxygenase system without having first been esterified. This may well happen when higher than normal concentrations of arachidonate are presented to the cell, as often occurs in experiments reported in the literature (15). The inhibitory effect of indomethacin on PMN phospholipase A2 is immediate and typical of a noncompetitive inhibitor. In contrast, inhibition of the prostaglandin synthetase system appears complex, involving both classical competition and an irreversible, time-dependent destruction of enzyme activity (5). We are aware of only one previous report of an inhibitory effect of indomethacin on phospholipid hydrolysis by phospholipase A2: At concentrations of 0.1-1 mM, more than 100 times the effective anti-inflammatory levels (5), indomethacin inhibited hydrolysis of egg lecithin by snake venom (Vipera ammodytes) phospholipase A2 (18). In this range of concentration, phospholipid hydrolysis was also inhibited under the conditions of our assays by two snake venom phospholipases A2 and by bee venom and pig pancreas phospholipases A2. However, at 50MM, a concentration that produces at least 70% inhibition of PMN phospholipase A2, indomethacin causes no detectable inhibition of these four phospholipases A2. It is not clear what accounts for the marked difference in sensitivity to indomethacin among enzymes that have many common features (19), such as molecular weight range and a high incidence of intramolecular S-S bridges. One reason may be that the sensitive PMN phospholipase A2 is tightly membrane-associated (8, 10), while all four insensitive phospholipases A2 are considered to be soluble enzymes. Conceivably, the molecular properties that promote membrane association are also those that result in susceptibility to indomethacin. However, it is unlikely that membrane association per se is an important factor in the susceptibility of PMN phospholipase A2 to inhibition by indomethacin, because inhibition is virtually the same with the membrane-associated phospholipase A2 activity in a crude homogenate (8, 10) and with a 500-fold-purified soluble phospholipase A2 preparation (Fig. 4 and Table 1). This indicates that the PMN phospholipase A2 is readily accessible to the inhibitor in the environment of the membrane and therefore suggests that the drug may be a useful tool in studies of the biological role of phospholipase A2 in leukocyte function (8-10, 17). 1. Vane, J. R. (1971) Nature New Biol. 231, 232-235. 2. Higgs, G. A., McCall, E. & Youlten, L. J. F. (1975) Br. J. Pharmacol. 53, 539-546. 3. Humes, J. L., Bonney, R. J., Pelus, L., Dahlgren, M. E., Sadowski, S. J., Kuehl, F. A., Jr. & Davies, P. (1977) Nature 269, 149-

151. 4. Smith, W. L. & Lands, W. E. M. (1971) J. Biol. Chem. 246,

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6700-6702. 5. Lands, W. E. M. & Rome, L. H. (1976) in Prostaglandins: Chemical and Biochemical Aspects, ed. Karim, S. M. M. (MTP, London) pp. 87-138. 6. Flower, R. J. (1974) Pharmacol. Rev. 26,33-65.

7. Elsbach, P. (1959) J. Exp. Med. 110,969-980. 8. Weiss, J., Franson, R., Beckerdite, S., Schmeidler, K. & Elsbach, P. (1975) J. Clin. Invest. 55,33-42. 9. Weiss, J. & Elsbach, P. (1977) Biochim. Blophys. Acta 466,

23-33. 10. Franson, R., Patriarca, P. & Elsbach, P. (1974) J. Lipid Res. 15, 380-388. 11. Mooney, C. & Elsbach, P. (1975) Infect. and Immun. 11, 1269-1277.

Proc. Nati. Acad. Sci. USA 75 (1978) 12. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917. 13. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 14. Hong, S. L. & Levine, L. (1976) Proc. Natl. Acad. Sci. USA 73, 1730-1734. 15. Borgeat, P., Hamberg, M. & Samuelson, B. (1976) J. Biol. Chem. 251,7816-7820. 16. Elsbach, P. & Farrow, S. (1969) Biochim. Biophys. Acta 176, 438-441. 17. Elsbach, P. (1972) Semin. Hematol. 9,227-239. 18. Bekemeier, H., Giessler, A. J. & Hirschelmann, R. (1974) Pol. J. Pharmacol. Pharm. 26,5-23. 19. Van den Bosch, H. (1974) Annu. Rev. Biochem. 43,243-277.

Low concentrations of indomethacin inhibit phospholipase A2 of rabbit polymorphonuclear leukocytes.

Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2955-2958, June 1978 Medical Sciences Low concentrations of indomethacin inhibit phospholipase A2 of...
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