Inflammation, Vol. 14, No. 4, 1990

CHARACTERIZATION OF EXTRACELLULAR PHOSPHOLIPASE A 2 (PLA2) ACTIVITY IN FLUID AND PERITONEAL CELLS FROM CASEIN-TREATED RATS T H E R E S A M. S T E V E N S , M I C H A E L M c G O W A N , J O H N G I A N N A R A S , and J A N E T S. KERR Medical Products Department E.1. du Pont de Nemours & Co., Inc. Experimental Station E400/4223 P. O. Box 80400 Wilmington, Delaware 19880-0400

Abstract--Extracellular phospholipase A2 activity (PLA2) found in the fluid and cells

of the peritoneal cavity of rats injected with casein is described. PLA2activities from both the fluid and cells require Ca2+ and have pH optima of 7. Acid-extraction increased PLA2 activity in the polymorphonuclear leukocyte (PMN) homogenates 20-fold but not the PLA2 activity in the extracellular fluid. Acid extraction also increased the sensitivity of the PLA2 activities to standard inhibitors. Since the PLA2 activities described in this model have characteristics similar to other inflammatory PLA2s, including human synovial fluid PLA2, casein stimulation should prove useful for testing potential inhibitors.

INTRODUCTION

Extracellular phospholipase A 2 (PLA2) activity is associated with many inflammatory processes, both local and systemic in man and animals (1-4). Extracellular PLA2 activity has been described in human synovial fluid from rheumatoid arthritic patients (1), lymphatic fluid in hypersensitivity reactions in sheep (2), casein-induced peritoneal inflammatory exudate in rats (3), and glycogeninduced peritoneal inflammatory exudate in rabbits (4). Although many investigators have described the release o f PLA2 from stimulated inflammatory cells such as polymorphonuclear leukocytes (PMNs) (5), macrophages (6), platelets (7), and synovial cells (8), it is not clear which cells in vivo are responsible for

389 0360-3997/90/0800-0389$06.00/0 9 I990 Plenum Publishing Corporation

390

Stevens et al.

the release o f this e n z y m e . T h e r e l e v a n c e o f e x t r a c e l l u l a r PLA2 activity in the in v i v o m o d e l s is also unclear. M a n y o f the P L A z s i n v o l v e d in the i n f l a m m a t o r y process h a v e h o m o l o g o u s N H 2 - t e r m i n a l s e q u e n c e s (9). C o m p a r i s o n with o t h e r k n o w n m a m m a l i a n P L A z s indicates that structural similarities exist b e t w e e n the c a s e i n - i n f l a m m a t o r y exudate PLA2 and that f r o m h u m a n s y n o v i a l fluid, rat platelets, and rabbit g l y c o g e n inflammatory exudate ( 1 0 - 1 2 ) . T h e NH2-terminal s e q u e n c e o f pancreatic PLAzs are not necessarily h o m o l o g o u s with these o t h e r m a m m a l i a n e n z y m e s (9). In the present study w e further c h a r a c t e r i z e d the e x t r a c e l l u l a r P L A 2 activity f r o m the casein-treated rat m o d e l o f inflammation. P L A 2 activities f r o m acid-extracted e x u d a t e fluid and f r o m peritoneal cells and the effects o f standard antiinflammatory agents on these activities w e r e e x a m i n e d . This m o d e l is simple and generates a large v o l u m e o f e x t r a c e l l u l a r PLA2 activity.

MATERIALS AND METHODS

Reagents. [3H]Oleic acid and Liquiscint were obtained from New England Nuclear Co. (Boston, Massachusetts). Bovine serum albumin (BSA), triethanolamine, sodium caseinate, dimethylsulfoxide (DMSO), and Crotalus adamanteus PLA~ were obtained from Sigma Chemical Co. (St. Louis, Missouri). M9CA media and HEPES buffer were obtained from Gibco (Grand Island, New York). All chemicals were reagent grade. Preparation ofE. coli Substrate. The membrane phospholipids of E. coli (strain HB101) were labeled with [3H]oleic acid according to the method of Patriarca et al. (13) as modified by Davidson et al. (14). The radiolabeled E. coli Were autoclaved at 120~ and 2.7 kg/cm2 for 15 rain. More than 90% of the incorporated label was in the 2-acyl position of membrane phospholipids as determined by treatment with PLA2 from Crotalus adamanteus. Extraction of E. coli Phospholipid and Determination of Specific Radioactivity. Radiolabeled E. coli cells were extracted according to Bligh and Dyer (15). The phospholipid obtained was resuspended in chloroform and quantitated on the basis of inorganic phosphate (Pi) (16). The specific radioactivity of this lipid was 5000 dpm/nmol of Pi, In our culture system 6.2 x 108 cells contained approximately 20 nmol of phospholipids; this agrees with previously reported values (14, 17). Preparation of PLA2from Casein-Treated Rats. Male Lew/CrlBr rats (Charles River Breeding Laboratories, Kingston, New York) weighing 200-250 g were used. Cells and fluid were isolated according to the method of Mackin et al. (18). Rats were injected intraperitoneally with 10 ml of 6% (w/v) sodium caseinate (or 0.9% saline for controls). Peritoneal cavities were lavaged with 10 ml heparinized saline 16 h after injection. The fluid was centrifuged at 500g on a Sorvall RC5C centrifuge (Du Pont, Newtown, Connecticut) for 10 miu at 4~ The supernataut was centrifuged again at 2000g. Using this method, approximately 15 ml of fluid per rat was obtained. The cell pellets were pooled and washed 3 • in Hanks' buffer (pH 7.2) at 4~ Cells obtained in this manner were more than 95 % PMNs as determined by Wright stain and light microscopic examination. In time course experiments the peritoneal cavities were lavaged from 4 to 48 h following casein injection. The percent of PMNs recovered varied with the time of lavage. Acid extraction of PLA2 activity from cells and fluid was performed according to the method of Fawzy et al. (19). Equal volumes of cells or fluid and ice-cold 0.36 N2SO 4 and 1.6 M NaC1 were mixed for 3 h at 4~ The mixtures were centrifuged at 20,000g (Sorvall RCSC centrifuge) for 20 rain. The supernatants were dialyzed overnight against two changes of 10 mM sodium

Extracellular Phospholipase A2 Activity in Rats

391

acetate, pH 4.5, and the dialysate was centrifuged at 20,000g for 20 min. The supernatants were designated "acid-extracted" cells or fluid. PLA 2 Assay Using E. coli Substrate. PLA 2 was assayed by a modified method of Rothhut et al. (20). [3H]oleic acid-labeled E. coli (50 ~1, 20 nmol Pi) was added to the PLA2 sample (10 #g protein in 100/xl of 1 : 100 dilution of peritoneal fluid with 0.9% saline) in 100 mM HEPES, pH 7.5, containing 0.2 mM calcium (standard PLA2 assay buffer), in a final volume of 250/xl. After 60 rain at 37~ in a shaking water bath, the samples were placed on ice. Hydrochloric acid (HC1) (100/xl of 2 N HCI) was added to stop the reaction and 100/xl of 2% BSA to trap the released [3H]oleic acid. The tubes were vortexed and then centrifuged at 1000g for 10 rain to pellet the bacteria. The supernatant, which contained the liberated fatty acids, was counted by liquid scintillation spectrometry (Hewlett-Packard 2200CA, Downers Grove, Illinois). To determine the percent recovery, the pelleted bacteria were hydrolyzed by adding 200 ~1 of 1 N NaOH, at 60~ for 30 min. HCI (200 ~1, 1 N) was added to neutralize the NaOH, and the fluid was counted as above. In similar experiments, pH, calcium, protein, and time of incubation at 37~ were varied. In experiments varying the pH, the enzyme was assayed as above with buffer substitutions of 100 mM sodium acetate, (pH 4-6), 100 mM Tris maleate (pH 6-8), 100 mM HEPES (pH 7-9), or 100 mM glycine (pH 9-10). Protein was determined by the Bradford method using bovine serum albumin as the standard (21). Effect of Inhibitors on PLA 2 Activity. For the inhibitor studies, manoalide, indomethacin, parabromophenacyl bromide (pBPB), piroxicam, or mepacrine were dissolved in DMSO and then brought to 1 mM final concentration in 100 mM HEPES, pH 7.5, containing calcium. The final concentration of DMSO was < 0.1%. Drugs were preincubated for 5 min at 4~ with the PLA2 sample (except pBPB, which was preincubated for 3 h) and then assayed as described above. Controls in which the enzyme or inhibitor were omitted were always included. Statistics. Results were reported as an average of triplicates _+ SEM and expressed as nanomoles of phospholipid (PL) hydrolyzed/per milligram of protein per hour. This was calculated as percent of total radioactivity and assumed that 6.2 x 108 E. coli cells equaled 20 nmot phospholipid. Results were compared by Student's t test (22). The level of statistical significance was taken as P -< 0.05.

RESULTS

Specific activities of crude cell homogenates and extracellular fluids were determined 16 h after casein injection and compared with the specific activities of acid extracts of the same samples (Table 1). Little (5 nmol PL/mg protein/ Table 1. PLA2 Activity in Total Cells or Extracellular Fluid from Casein-Treated Ratg' Activity (nmol PL hydrolyzed/mg protein/hr) Extracellular fluid

Cells

Acid-extracted fluid

Acid-extracted cells

246 • 40

4.5 _+ 1.4

200 + 1.0

100 + 8.0*

~'Cells and extracellular fluid were isolated as described in Materials and Methods, 16 h after 6% casein injection. Both the fluid and the cell sonicates were acid extracted as described. PLA2 activity was determined using E. coli as the substrate. Data are expressed as mean +SEM (N = 4). *P < 0.005 compared with corresponding unextracted samples.

392

Stevens et al.

7-

A.

6~

5-

"5 E ~=

4-

,#

"6 "6 < 1 0 0

50

250-

"~-

1 O0

150 200 Protein (/zg)

250

300

i 350

B,

200-

150"5 100.6 < 52

50

-

0

1'o

4 pH

Fig. 1. Characteristics of PLA 2 activity in acid extracts of extracellular peritoneal fluid or cells from casein-treated rats using E. coli as substrate. (A) Protein (N = 2); (B) pH (N = 3); (C) calcium (N = 3); (D) time of incubation (N = 2). Solid line: extracellular fluid; dashed line: cells.

h) PLA 2 activity was present in whole cell homogenate compared to the PLA2 activity from extracellular fluid (246 nmol PL/mg protein/h). PLA2 activity in cell acid extracts was 20 times higher (100 nmol/mg protein/h) than in the whole cell homogenate. Surprisingly, PLA 2 activity in extracellular fluid was not enhanced by acid extraction. Characteristics of PLA2 Activity. The characteristics of PLA2 activity in acid-extracted peritoneal fluid and cells of casein-treated rats are shown in Figure 1. Figure 1A shows the protein dilution curves of PLA2 activities. The levels of PLA2 activity in acid extracts of cells from casein-treated rats increased from 0.016 nmol PL/h at 0.4/zg protein to 2.48 nmol PL/h at 320 tzg protein. There was threefold higher PLA2 activity in acid extracts of fluid (6.1 nmol PL/h) at 320 tzg protein than in acid extracts of cells. PLA2 activities were linear

Extracellular

Phospholipase

Az

200-

C,

Activity in R a t s

393

g "5 ft. 1 5 0 -

13. 100v > "5 < 0

50-

if) , , , , . . . . . . SS

o

5

1 EDTA

5OO

H,q ~ iHl,lq r rlllllq , IlH,,q , I1;1,, I 0.01 0.1 1 10 100 (mM) Calcium

D.

400

300

eoo "6 9~

lOO

< i

i

50

1 O0

Fig.

i

i

150 200 Time (min)

i

I

250

300

1. Continued.

with respect to protein up to 10/xg in acid extracts from both cells and fluid. In subsequent assays, 10 p.g protein was used. Because many PLA2s have a pH optimum in the neutral range, the effect of pH on the PLA2 activity in acid extracts of cells and fluid was examined (Figure 1B). The peak of PLA2 activity was between 191 and 200 nmol PL/mg protein/h at pH 7-9 (Figure 1B) from acid-extracted fluid. In acid extracts of cells, the peak PLA2 activity was also broad and in the neutral range, pH 7-10 (124-127 nmol PL/mg protein/h). PLA 2 activity in unextracted peritoneal fluid also had a broad neutral pH optimum (data not shown). Many PLA2s require calcium for activity. PLA2 activity from acid-extracted cells and extracellular fluid was highest at 0.2 mM calcium (107 and 146 nmol PL/mg protein/h, respectively) (Figure 1C). EDTA (1 and

394

Stevens et al.

5 mM) inhibited these PLA 2 activities. Using a 100 mM HEPES buffer at pH 7.5 containing 0.2 mM calcium, PLA 2 activity as a function of time was determined. Both the cellular and extracellular PLA2 activities were linear up to 30 min (Figure 1D) (63 and 202 nmol PL/mg protein/h, respectively), at which time the rate of hydrolysis began to plateau. Cell Influx and PLA2 Activity. To determine whether the PLA2 activity in the extracellular fluid correlated with the influx of cells into the peritoneal cavity, cells and fluid were collected at 0, 5.5, 16, 24, and 48 h following casein injection. The total number of cells in the peritoneal cavity increased with time after casein injection, reaching a maximum of 1.33 x 10 9 cells at 48 hr (Figure 2). Differential analysis of the cell samples showed that PMNs were greater than 95% at 5.5, 16, and 24 h. However, at 48 h the percent of total cells that were PMNs was reduced to only 53 %. Forty-seven percent of the cells were monocytes at 48 h. The specific activity of PLA 2 in the extracellular fluid also significantly increased from 79 nmol PL hydrolyzed/mg protein/h at 5.5 h after casein-treatment up to 165 nmol PL hydrolyzed/mg protein/h at 24 h after casein-treatment. PLA 2 activity decreased in the 48-h casein-treated rats to 134 nmol PL hydrolyzed/mg protein/h. Thus, although the total number of cells 160g" 1 4 0 -

6,.

12o

100.~ 8 0 E= 6 0 z 4020o

i

Saline

I 5.5 hr

I 16 hr

Casein Casein

w 200-

I 2 4 hr

I 4 8 hr

Casein

Casein

B.

~ 150-

uJ

~

5oE

o

I" Saline

N I

I

I

I

5.5 hr

16 hr

24 hr

48 hr

Casein

Casein

Casein

Casein

Fig. 2. Time course of cell infiltration into the peritoneal cavity of rats after casein injection. Total cells and fluid were isolated from the peritoneal cavity at 5.5, 16, 24, or 48 h after casein injection. Total cells, PMNs, and monocytes were determined (A). Hatched bars: total; stippled bars: PMNs; open bars: monocytes. PLA2 activity in the extracellular fluid was measured (B). *P < 0.05 compared to respective saline controls; +P < 0.05 compared to 5.5, 16, and 48 h.

Extracellular Phospholipase A2 Activity in Rats

395

increased up to 48 h, the peak of extracellular PLA2 activity per milligram of protein in fluid at 24 h roughly paralleled the percentage of total cells that were PMNs in the peritoneal cavity. Effect o f Inhibitors on PLA 2 Activities. The effects of several antiinflammatory agents on PLA2 activity from the various fractions were examined. However, the inhibitory effects of these agents on the hydrolysis of E. coli substrate by crude cell homogenates was not determined because of the low PLA2 activity (5 nmol PL/mg/protein/h). Of the agents tested, manoalide had the greatest inhibitory activity on all three enzyme sources. Manoalide irreversibly inactivates cobra venom PLA2 through modification of the lysine residues (23). The ICso for manoalide in the extracellular fluid was 60/xM, 41 txM for acid-extracted fluid, and 19/xM for acid extracts of cells. p-Bromophenacyl bromide (pBPB) is an acylating agent that forms adducts with histidine residues in PLA2 (24). The ICso of pBPB was 3 mM for extracellular fluid PLA2 activity. Acid extraction of the extracellular fluid resulted in a 10-fold decrease in the IC5o to 330 ~M. In acid extracts of cell homogenates, pBPB had an ICs0 of 130/xM. Inhibition of PLA 2 by mepacrine (an acridine derivative) has been reported (25-27). In the present study, mepacrine was more effective against the PLA2 activity in acid-extracted fluid (ICso -- 170 IzM) than in crude extracellular fluid (420 ~M). Mepacrine also inhibited PLA 2 activity in acid extracts of cells (IC50 = 750/~M). The nonsteroidal antiinflammatory agent indomethacin inhibited the extracellular fluid and acid extracts of fluid and cells with ICs0 values of 1300, 900, and 560/zM, respectively.

DISCUSSION PLA 2 catalyzes the deacylation of the fatty acids from the sn-2 position of phospholipids. One of these fatty acids, arachidonic acid, can serve as a substrate for the generation of inflammatory lipid mediators such as eicosanoids; the lysophospholipid can serve as the precursor for platelet-activating factor. Inhibition of PLA2 activity offers an attractive therapeutic approach to the treatment of inflammatory diseases. Several investigators have described the selective release of extracellular PLA2 activity from cells involved in the inflammatory process (5-8). Many of these PLA2s, including the casein-stimulated PLA2, have protein sequence homologies (9). This paper describes a useful model to study this PLA2 activity from an inflammatory site. The characteristics of PLA2 activity from casein-treated rats are similar to those of other extracellular PLA2s (1-4, 28, 29). More importantly, this enzyme

396

Stevens et al.

activity has the same characteristics as those associated with the cells involved in the inflammatory process. The PLA2 activities described here, before and after acid extraction, have broad neutral (7-9) pH optima, are stimulated by low levels of calcium (0.2 raM), and are inhibited by EDTA. PLA2 activities from both the acid-extracted and non-acid-extracted extracellular fluid are similar, but not identical to those described by Chang et al. (3). The activities described here have broader pH optima (pH 7-9 rather than 9) and a sharper sensitivity to calcium. Acid extraction results in a 20-fold purification of PLA: activity from peritoneal cells. This finding is similar to that of Forst et al. (12), who also found a 20-fold purification of PLA2 from PMN acid extracts. In contrast, acid extraction does not enhance the PLA 2 activity in extracellular fluid. The potency of the tested inhibitors increased with acid extraction; the acid-extracted fluid had lower ICs0 values than the crude extracellular fluid. Manoalide and pBPB, both alkylating agents, are expected to have lower IC50 values because the acid treatment removes non-PLA 2 protein. However, the decreases in IC5o values with mepacrine and indomethacin are not as easily explained, except that the structure of the enzyme also may be affected by acid extraction. Although Chang et al. observed partial inhibition of crude extracellular fluid PLA2 activity by pBPB (28), the present study is the first report of IC50 values of crude fluid using all of these inhibitors. It is also the first report of increased efficacy of the inhibitors in acid extracts of extracellular fluid. The observation that these inhibitors (except mepacrine) have similar effects on the PLA2s from acid-extracted cells and fluid suggests that these enzymes may be related. The time course of extracellular PLA 2 activity and the influx of inflammatory cells (PMNs, monocytes) into the peritoneal cavity were examined to determine the correlation between PLA: activity and cell influx. The data suggest that PLA2 activity correlates with increases in PMN influx. At 0 h (saline control) the PLA2 activity is 0.1 nmol PL/mg protein/h, and the cell population consists of 98% monocytes, 2% PMNs. The PLA2 activity increases with casein treatment at 24 h to a peak of 165 nmol PL/mg protein/h, at which time 95 % of the cells are PMNs. This time course is slightly longer than Chang and coworkers reported (3); they found maximal PLA2 activity and PMN influx at 15 h. At 48 h PLA 2 activity decreases to 134 nmol PL/mg protein/h, and only 53 % of the cells present are PMNs. These results suggest that the influx of PLA2 activity in the extracellular fluid is PMN-associated at 24 h. At 48 h, the total number of PMNs is actually increased, but comprise a lower percentage of total recoverable cells. These findings are in contrast to those of Chang and associates (3), who showed at 48 h after casein injection total peritoneal cells, PMNs, and PLA2 activity all decreased. The reason for these differences is not apparent but may be related to the volume and concentration of casein that was used to elicit the inflammatory response.

Extracellular Phospholipase A2 Activity in Rats

397

There are several potential sources for the PLA 2 activity found in the peritoneal cavity after casein injection. One possibility is that the stimulated PMNs release PLA2 into the peritoneal cavity. We, as well as Chang et al. (3), have shown that the characteristics of the extracellular PLA2 are similar to those of the cell-associated PLA2. On the other hand, many PLA2s have similar characteristics (29, 30). Another possible explanation was suggested by Forst et al. (31): that the PLA 2 in the blood is carried (i.e., piggy-back) by the PMNs migrating into the cavity. Although we did not measure the platelet population, we cannot role out the third possible explanation that the PLA2 activity is derived from rat platelets either from the blood or in the peritoneal cavity. Rat platelets are known to release extracellular PLA2 in inflamed sites. The rat platelet-associated PLA2 is the same as the released enzyme and is similar to the PMNassociated PLA 2 (3, 7, 33). The extracellular PLA 2 from human synovial fluids in rheumatoid arthritis is similar to the rat platelet PLA2 and casein-elicited extracetlular PLA2, as determined by amino acid composition and partial NH2-terminal amino acid sequence (10, 11, 32). Since the PLA2 activity from casein-induced inflammation is similar to the human extracellular synovial enzyme, and since the rat enzyme activity is readily accessible, this model could serve as a useful source for PLA 2 activity in testing for potential inhibitors of PLA 2.

REFERENCES 1. PRUZANSKI,W., P. VADAS,E. STEFANSKI,and M. B. UROWITZ. 1985. Phospholipase A~ activ-

2. 3. 4.

5.

6.

7.

8.

ity in sera and synovial fluids in rheumatoid arthritis and osteoarthritis. Its possible role as a proinflammatory enzyme. J. Rheumatol. 12:21t-216. VADAS,P., and J. HAY. 1982. The appearance and significance of phospholipase A 2 in lymph draining tuberculin reactions. Am. J. Pathol. 107:285-291. CHANG, H. W., I. KUDO, S. HARA, K. KARASAWA,and K. INOUE. 1986. Extracellular phospholipase A2 activity in peritoneal cavity of casein-treated rats. J. Biochem. 100:109%1102. FRANSON, R., C. DOBROW, J. WEISS, P. ELSBACH, and W. WEGUCKI. 1978. Isolation and characterization of a phospholipase Aa from an inflammatory exudate. J. Lipid Res. 19"1823. TRAYNOR,J. R., and S. AUTm. 1981. Phospholipase A2 activity of lysosomal origin secreted by polymorphonuclear leukocytes during phagocytosis or on treatment with calcium. Biochim. Biophys. Acta 665:571-577. W~GnTMAN,P. D., D. M. DAnLGREN, E. P. DAVIES, P., and R. J. BONNEu 1981. The selective release of phospholipase A 2 by resident mouse peritoneal macrophages. Biochem. J. 200:441-444. HORIGOME,K., M. HAYAKAWA,V. INOUE, and S. NOJIMA. 1987. SeLective release of PLA2 and lysophosphatidylserine-specific lysophospholipase from rat platelets. J. Biochem. 101:5362. PRUZANSKI,W., P. VADAS, J. KIM, H. JACOBS, and E. STEEANSKI. 1988. Phospholipase A2 associated with synovial fluid cells. J. Rheumatol. 15:791-794.

398

Stevens et al.

9. LAI, C., and K. WADA. 1988. Phospholipase A2 from human synovial fluid: Purification and structural homology to the placental enzyme. Biochem. Biophys. Res. Commun. 157:488-493. 10. HARA, S., I. KUDO, K. MATSUTA,T. MIYAMOTO,and K. INOUE. 1988. Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purification from rheumatoid synovial fluid. J. Biochem. 104:326-328. 11. HAYAKAWA,M., I. KUDO, M. TOMITA, and K. INOUE. 1988. Purification and characterization of membrane-bound phospholipase A2 from rat platelets. J. Biochem. 103:263-266. 12. FORST, S., J. WEISS, P. ELSBACH,J. M. MARAGANORE,I. REARDON,and R. L. HEINRIKSON. 1986. Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate. Biochemistry 25:8381-8385. 13. PATRIARCA,P., S. BECKERDITE,and P. ELSBACH.1972. Phospholipases and phospholipid turnover in Escherichia coli spheroplasts. Biochim. Biophys. Acta 260:593-600. 14. DAVIDSON,F. F., E. A. DENNIS, M. POWELL, and J. R. GLENNEY, JR. 1987. Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids. J. Biol. Chem. 262:1698-1705. 15. BLIGH,E. G., and W. J. DYER. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 16. BARTLETT,G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466468. 17. FRANSON,R., P. PATRIARCA,and P. ELSBACH. 1974. Phospholipid metabolism by phagocytic cells. Phospholipase A2 associated with rabbit polymorphonuclear leukocyte granules. J. Lipid Res. 15:380-388. 18. MACKIN, W. M., S. M. RAKICH, and C. L. MARSHALL. 1986. Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug. Biochem. Pharmacol. 35:917-922. 19. FAWZY, A. A., R. DOBROW, and R. C. FRANSON. 1987. Modulation of phospholipase A2 activity in human synovial fluid by cations. Inflammation 11:389-400. 20. ROTHHUT, B., F. RUSSO-MARIE,J. WOOD, M. DIROSA, and F. J. FLOWER. 1983. Further characterization of the glucocorticoid-induced antiphospholipase protein "renocortin." Biochem. Biophys. Res. Commun. 117:878-884. 21. BRADFORD,M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 22. SNEDECOR, G. W. 1964. Sampling from normally distributed population. In Statistical Methods, 5th ed. G.W. Snedecor and W.C. Cochran, editors. Iowa State University Press, Ames. 45. 23. REYNOLDS,L. J., B. MORGAN, G. HITE, E. MIHELICH, and E. DENNIS. 1988. Phospholipase A2 inhibition and modification by manoalogue. J. Am. Chem. Soc. 110:5172-5177. 24. ROBERTS,M. F., R. DEEMS, T. MINCEV, and E. DENNIS. 1977. Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). J, Biol. Chem. 252:2405-2411. 25. FLOWER,R. J., and G. BLACKWELL.1976. The importance of phospholipase A2 in prostaglandin biosynthesis. Biochem. Pharmacol. 25:285-291. 26. LAPETINA,E. G., M. B1LLAH,and P. CDATRACASAS.1981. The initial action of thrombin on platelets. J. Biol. Chem. 256:5037-5040. 27. HIRATA, F., B. CORCORAN, K. VENKATASUBRAMANIAN, E. SCHIFFMANN, and J. AXELROD. 1979. Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes. Proc. Natl. Acad. Sci. U.S.A. 76:2640-2643. 28. CHANG, H., I. KUDO, M. TOMITA, and K. INOUE. 1987. Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rats. J. Biochem. 102:147-154.

Extracellular Phospholipase A 2 Activity in Rats

399

29. WAITE, M. 1987. Phospholipase A2 of mammalian cells. In Handbook of Lipid Research, Vol. 5, The Phospholipase. D. J. Hanahan, editor. Plenum Press, New York. 111-133. 30. WAITE, M., H. GRIFFIN, and C. R. FRANSON. 1976. The phospholipases of iysosomes. In Lysosomes in Biology and Pathology, Vol. 5. A. Neuberger and E.I. Tatus, editors. ElsevierNorth Holland, New York. 257-305. 31. FORST, S., J. WEISS, and P. ELSBACH. 1986. Properties of an inflammatory exudate phospholipase A 2 that degrades the phospholipids of E. coli during phagocytosis. Clin. Res. 34:724A. 32. HARA, S., I. KUDO, H. CHANG,K. MATSUTA,T. M1YAMOTO,and K. INOUE. 1989. Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis. J. Biochem. 105:395-399. 33. MIZUSHIMA, H., I. KUDO, K. HORIGOME, M. MURAKAMI, M. HAYAKAWA, D.-K. KIM, E. KONDO, M. TOMITA, and K. INOUE. 1990. Purification of rabbit platelet secretory phospholipase A 2 and its characteristics. J. Biochem. 105:520-525.