Possible role of phospholipase A2 in triggering histamine secretion from human basophils in vitro

CLINICAL

IMMUNOLOGY

Possible

AND

Role of Phospholipase Secretion from Human GIANNI

The Johns

20, 231-239

IMMUNOPATHOLOCY

Hopkins

Uni~vrsity

(1981)

AZ in Triggering Histamine Basophils in Vitr~‘,~

MARONE,~ ANNE KAGEY-SOBOTKA, LAWRENCE M. LICHTENSTEIN~ School Immunology,

of Medicine, Baltimore,

Received

January

Department Maryland

AND

of Medicine. 21239

Division

of Clinical

12. 1981

We have investigated the possibility that phospholipase A, (PLA,) activation plays a role in immediate hypersensitivity reactions. Two agents, p-bromophenacyl bromide (BPB) and mepacrine, which inhibit PLA, activity in several tissues, cause a dosedependent inhibition of histamine release from human basophils induced by several immunologic (i.e., antigen and anti-IgE) and nonimmunologic (i.e., formyl-methioninecontaining peptide [(f-met peptide)] and the Ca *+ ionophore, A23187) stimuli. The inhibitory effect of BPB on histamine release is irreversible and extremely rapid. BPB is inhibitory in the whole reaction as well as in the first and second stage of antigen- and in the extracelanti-IgE-induced histamine release. Increasing the Ca’+ concentrations lular medium partially blocks the inhibitory effect of BPB. BPB, however, is not a competitive antagonist of the effect of Ca?’ suggesting that the two agents act on distinct enzymatic sites. The inhibitory effect of BPB in the presence of exogenous arachidonic acid (AA) was less marked than in the absence of AA. These data suggest that the activation of cell surface receptors and/or the intracellular translocation of C3’ by the ionophore A23187 activates a basophil PLAZ which plays an essential role in the control of the release of preformed mediators.

INTRODUCTION

The activation of human basophils, by either immunologic or nonimmunologic stimuli, initiates a complex sequence of biochemical reactions culminating in the release and/or de nova synthesis of inflammatory mediators (1). Although the available evidence indicates that cyclic adenosine 3’,5’-monophosphate participates in both the activation and the modulation of the release process (2-4), little is known of the specific biochemical steps involved in basophil secretion. We recently reported that 5,8,11,14eicosatetraynoic acid (ETYA),” which nonselectively blocks both the cyclooxygenase and lipoxygenase pathways of arachidonic ’ This work was supported in part by Grants AI-07290 and HL-23586 from the National Institute of Allergy and Infectious Disease and the Heart, Lung and Blood Institute, National Institutes of Health. ? Publication No. 429, O’Neill Research Laboratories, The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Md. 21239. 3 Supported in part by a Grant from the CNR (Rome, Italy). Present address: IJniversity of Naples II School of Medicine, Department of Medicine, Via S. Pansini 5 Cappella Cangiani, 80131 Naples. Italy. ’ Address reprint requests to Dr. Lawrence M. Lichtenstein, The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Md. 21239. 3 Abbreviations used: CAMP, Adenosine 3’.5’-monophosphate: Pipes. piperazine-N-N’-bis(2ethanesulfonic acid); AgE. antigen E; Anti-IgE, goat anti-human IgE; AA, arachidonic acid: PLA,. phospholipase A,: ETYA. 5,8.11.14-eicosatetraynoic acid: BPB, p-bromophenacyl bromide: f-met peptide, formyl-L-methionine-r-leucine-t.-phenylalanine. 231 0090-1229/81/080231-09$01.00/O Copyright All rights

@ 1981 by Academic Press. Inc. of reproduction in any form reserved.

232

MARONE,

KAGEY-SOBOTKA,

AND

LICHTE~NSTEln.

acid (AA) metabolism (5), caused a dose-dependent inhibition of antigen-induced histamine release from human basophils (6). In contrast. nonsteroidal antiinflammatory drugs. at concentrations which block only the cyclooxygenase pathway, induced a significant enhancement of antigen-induced histamine release (6). These observations suggested that a metabolitets) of AA. generated via a lipoxygenase pathway. is involved in IgE-mediated histamine secretion. This hypothesis has been strengthened through the use of a more specific inhibitor of lipoxygenase activity. 5,8,11-eicosatriynoic acid. which inhibited the basophil histamine release induced by antigen, anti-IgE, the f-met peptide. and the Ca”+ ionophore. A23187 (7). Other studies indicate that an increase in phospholipid turnover is associated with the secretion of histamine from rat mast cells and basophil leukemia cells (8, 9). Involvement of phospholipase A, (PLA,) in the release process was suggested by the work of Uvnas and his colleagues in the 1950s (IO). Since PLA, activation generates AA and plays a pivotal role in phospholipid turnover. it seemed reasonable to investigate the role of this enzyme in the secretion of basophil histamine. MATERIALS

AND METHODS

Reagcrzrs. Piperazine-N-N’-bis(2-ethanesulfonic acid) (Pipes). mepacrine, and arachidonic acid (AA) were purchased from Sigma Chemical Company. St. Louis. Missouri. Human serum albumin was obtained from Miles Laboratories. Inc.. Elkhart, Indiana. ETYA was obtained and prepared as previously described (6). p-Bromophenacyl bromide (BPB) was obtained from Aldrich Chemical Company. Milwaukee, Wisconsin. BPB (5 x IO-’ M) was dissolved in acetone in the dark at 22°C. A fresh preparation was made immediately before use. Acetone. at the concentrations used, had no effect on histamine release. Antigens. mti-IRE. c,trlcVutn iowplzow .4.?3/87. trrrcl l&et pcpticlr. Histamine release was initiated by challenge with the ragweed antigen E ( 1 I ). Rye Group I ( 12), anti-IgE (a-IgE) (13), the Ca” ionophore. A23 187, or the formylated tripeptide, formyl-methionine-leucine-phenylalanine (f-met-peptide) ( 14). These reagents were kindly supplied by Dr. T. P. King, Rockefeller University. New York, New York, Dr. K. Ishizaka, and Dr. D. Marsh, Johns Hopkins University. Baltimore, Maryland, Dr. R. Hamill, Lilly Research Laboratories, Indianapolis. Indiana, and Dr. E. Schiffmann, National Institutes of Health. Bethesda. Maryland, respectively. A23187 was dissolved in dimethylsulfoxide (DMSO) (2 mgiml). At the concentrations used DMSO alone had no effect on the release process ( 15). Buglers. (a) PA, which contains 25 mM Pipes, I10 mM NaCI, 5 mh! KCl, and 0.01% human serum albumin, was used to wash the leukocytes: (b) PACM. used for release studies, contained, in addition to the above. 0.6 mM CaCI,.6H,O and 1.0 mM MgCI.,. The buffers were adjusted to pH 7.3 before use t 16). Leukocyte dor7ors. Venous blood was obtained from normal and allergic adult volunteers who had given informed consent. The donors had no history of aspirin idiosyncracy and were not symptomatic when their blood was used. The donors had not taken any systemic medications for at least 48 hr nor any antiinflammatory drugs for at least 1 week before venesection.

PHOSPHOLIPASE

A, AND BASOPHIL

HISTAMINE

RELEASE

233

Histamine release. Blood was drawn into dextran-EDTA, allowed to sediment for 90 min, washed twice in PA, and the leukocytes suspended in PACM prior to challenge (6). Individual assay tubes usually contained the leukocytes from 0.5 ml of whole peripheral blood. BPB and mepacrine were preincubated with cells for 2 and 20 min, respectively. The release reaction was then initiated by addition of the various stimuli and allowed to proceed for 45 min at 37°C. The total reaction volume was 1.0 ml. All experiments were carried out in duplicate or triplicate using the cells from three or more different donors, in separate assays, for each experimental design. When the two stages of histamine release were studied (16), cells were incubated for 2 min with antigen, with or without drug, in the absence of Ca*+ and Mg*+, washed twice in PA, and resuspended in PACM for release to occur. Following the release reaction, the leukocytes were removed by centrifugation (1OOOg for 1 min), and the supernatants assayed for histamine content. The difference between replicate histamine measurements was less than 5%. Aliquots of cells in the same total volume were lysed with 2% perchloric acid (HClOJ to evaluate total histamine. Each study included control tubes containing cells to which no stimulus was added. Spontaneous histamine release (Z!-5% of the total histamine) was subtracted from all values and the percentage histamine release calculated. With the exception of mepacrine (see below), none of the drugs used interfered with the histamine assay. Histamine was measured using an automated fluorometric technique (17, 18). When mepacrine, which itself fluoresces, was used the incubation was performed as usual following which the cell pellets were washed four times with 2 ml of ice-cold PA, then lysed with HCIOd, and assayed for residual histamine.

b

BPB(Ml

FIG. 1. (a) Dose-response curves of inhibition by BPB of antigen and anti-IgE-induced histamine release. The mean ?SEM of 5 to 11 experiments is shown for each BPB concentration. The range of histamine release for antigen was 42-76%. for anti-IgE, 44483%. (b) Dose-response curves of inhibition by BPB of ionophore (A23187)- and f-met peptide-induced histamine release. The mean k SEM of 3 to 6 experiments is shown for each experimental point. The range of histamine release for ionophore (0.5 &ml) was 38-63%, for f-met peptide, 47-52%.

234

MARONE,

KAGEY-SOBOTKA,

AND

LICHTENSTEIN

RESULTS

p-Bromophenacyl bromide (BPB), an inhibitor of PLA.,, has been shown to alkylate the imidazole side chain of the histidine in position 53, at the active site of purified preparations of this enzyme (19-21). We tested the effect of BPB on histamine release from human basophils induced by immunologic (antigen and anti-IgE) and nonimmunologic (f-met peptide and Ca’)+ ionophore. A23187) stimuli. BPB caused a dose-dependent inhibition of histamine release induced by both immunologic stimuli (Fig. la): measurable inhibition occurred at lOpi M, 50% inhibition at about lo-” M. and virtually complete suppression of release was observed at lo-” M. Similar results were obtained when the cells were treated with BPB and challenged with the f-met peptide or the Ca’- ionophore. A23187 (Fig. lb). The effect of BPB on each releasing agent studied was quite reproducible using the basophils of at least three donors for each stimulus. The inhibition caused by BPB occurred over a wide concentration range of antigen (Fig. 2a)- and anti-IgE (Fig. 2b)-induced release, including those which caused antigen and antiIgE excess inhibition. Mepacrine has been shown to inhibit PL.A, activity in several systems (22- 24). It also inhibited the histamine release induced by antigen, anti-IgE, f-met peptide, and the Ca”- ionophore (Table 1). The inhibition was dose dependent with a 50% effect occurring between 30 and 100 pM. Since BPB is a more specific antagonist of PLA, than is mepacrine (25). additional studies were limited to the former. Sfcrtlic~s m

flrc Mwlrtrrrisnz

of’ l~l~libitioti

c!f‘ H~J fumim*

Rslctr.w

hi, BPH

The inactivation of purified PLA? by BPB has been shown to be irreversible (26): this was also the case with respect to the inhibitory effect of BPB on histamine release. A similar inhibition dose-response curve was generated when cells were incubated with various concentrations of BPB for 10 min and then

, _ .. .

t IO5 a

10..

-7 ~~~~ ~ id' ANTlGEN EoJg/mi~

-7 IO2

. 11103

In b

1 "I,> %

: oi

l

3riO'M

,

.*; !.102 Ahi

,rlC ' IqELygml.

FIG. 7. Dose-response curves of (a) antigen E- and (b) anti-IgE-induced histamine release. alone (closed symbols) and in the presence of three concentrations of BPB (open symbols). Inhibition of release by both stimuli was observed with each BPB concentration throughout the entire dose-resoonse curve.

A,

PHOSPHOLIPASE

AND

BASOPHIL

TABLE THE

EFFECT

OF MEPACRINE

Stimulus

Experiment: No drug Mepacrine, Mepacrine. Mepacrine, Mepacrine. Mepacrine,

3 1 3 1 3

x

x x

x x

10m6M lo-” M lo-” M lo-'M lo-’ M

HISTAMINE

235

RELEASE

1

ON HISTAMINE

RELEASE

FROM

HUMAN

BASOPHILS”

Antigen

Anti-IgE

f-met Peptide

Ca?’ ionophore

(% HR)

(5% HR)

(9%HR)

(% HR)

1

2

1

2

1

2

1

75 71 62 57 23 0

72 72 67 60 37 0

68 32 26 2

68 63 57 52 25 6

49 42 23 8 0 0

39 27 22 8 0 0

56 46 38 34 18 0

” Cells were incubated with mepacrine for 20 min at 37°C before the addition of the various stimuli. Histamine release (HR) was allowed to proceed for an additional 45 min at 37°C.

washed, or not washed, prior to antigen challenge (Fig. 3). BPB also inhibited histamine release equally well whether added 30 min or 1 min before the releasing agent (data not shown). BPB not only acts rapidly but exerts its effect at any time during the release process. In the experiment shown in Fig. 4, the kinetics of anti-IgE-induced histamine release were established by adding, at the times indicated, 3 mM EDTA (final concentration), centrifuging the cells rapidly (‘1 min), and obtaining the supernatants. Separate aliquots were taken at the same time intervals and incubated at 37°C in either 3 mM EDTA or lo-” M BPB until the termination of the experiment. The addition of BPB at any time during the reaction led to prompt cessation of histamine release. As previously reported, the release process is also stopped immediately upon chelation of Ca2+ by EDTA (27).

FIG. 3. The irreversibility of the inhibition of histamine release by BPB. Two sets of cells were preincubated for 10 min at 37°C with 3 x IO-“- 1 x 10’ M BPB or buffer (controls). One set was then challenged with anti-IgE (5 x lo-? &ml) and release allowed to proceed for 45 min at 37°C (0). The second set of cells was washed twice with PA, resuspended in PACM, and challenged with anti-IgE and release allowed to proceed as above (0). Histamine release in the absence of BPB was 58%.

236

MARONE,

KAGEY-SOBOTKA,

AND

LICHTENSTEIN

. EDTA + Cenfrlfugotwn

0

5

.

IO

15

20

33

40

50

TIME(mtnl

FIG. 4. The effects of BPB and EDTA on anti-IgE-induced anti-IgE is shown (0). Addition of IO-” M BPB (0) or 3 mM release process immediately stopped the reaction.

histamine release. The kinetic curve EDTA (A) at any time (arrows) during

for the

Additionally, BPB inhibited equally well when added to the first or second stages of the release reaction (data not shown). These findings suggest that PLA, activation is ongoing during the entire process of histamine release. Interaction

het,zlern Ctr” and PLA,

Ca2+ is required for both histamine release (27) and PLA, activation (26, 28). Although Ca2+ and BPB do not bind to the identical site on this enzyme (19, 28), it has been proposed that the ion might protect PLA, against inactivation by this compound (28). We, therefore, examined the possible interaction between Ca” and BPB. Three experiments showing inhibition of antigen-induced histamine release by BPB (0.6-2 PM) at Ca2+ concentrations ranging from 0.3 to 6.0 m&I are presented in Table 2. Increasing the concentration of Ca’+ in the extracellular fluid caused reductions in the inhibitory effect of BPB. Experiments exemplified by those shown in Table 2 were used to construct Schild plots (29); it was found that TABLE THE

Er-~tcr Expt

OF Ca2-

I %HR

2

Expt ___~ C/r’I

-

I.2

1.0

-

1.0

0.6

CaL- (mM) 6.0 2.5 1.0 0.3

X6 81 78 66

31 35 50 89

27 31 36 48

91 81 72 59

15 21 39 44

x 6 6 29

versus

the control

69 70 62 51

3 5 I

!‘+ H R

BPB (/r/k’)

(’ Control histamine release. ” Inhibition: the percentage was calculated

BY BPB

ON INHIBIIION

Expt

Q 1I,

“rHR”

2

CONCENTRATION

2.0

1.0

0.6

77 84 92 100

48 64 74 78

13 46 37 41

HR at the same Ca’.

concentrations.

A, AND BASOPHIL

PHOSPHOLIPASE

HISTAMINE

RELEASE

237

the slopes were significantly different from 1.0 (P < 0.05; Student’s t test) [data not shown]. This suggests that BPB and Ca2+ do not interact with the same binding site and is compatible with previous results obtained using the purified enzyme in cell-free systems (19, 28). The Effects of Arachidonic Acid (AA)

It is reasonable to suggest that the role of PLA, in histamine release is to generate AA and that BPB is inhibitory because it limits the availability of this product. We have reported that exogenous AA ( 10p6- lop5 M) enhances antigeninduced histamine release from human basophils but that higher concentrations inhibit the response (6). The AA analog, 5,8,11,14eicosatetraynoic acid (ETYA) causes a dose-dependent inhibition of antigen-induced histamine release (6). As shown in Table 3, arachidonic acid potentiates the inhibitory effects of ETYA. In the two experiments shown, 3 x 1OV M AA, which itself potentiated release by antigen E, caused marked increases in inhibition by ETYA. ETYA (3 pg/ml) alone had little effect on release, but in the presence of AA release was reduced by 40 to 80%. ETYA (5 pg/ml) alone was inhibitory; again this effect was augmented by exogenous AA. These data suggest that AA adds to the endogenous accumulation of the arachidonate (due to the block of the AA cascade by ETYA) leading to intracellular concentrations of AA which are in the inhibitory range. The interaction between AA and the PLA, inhibitor, BPB, was next examined as shown in Table 4. AA (10e6 M) partially reversed BPB inhibition in each of three experiments. Less reversal occurred at the highest BPB concentration tested (11-25010) whereas with lo-’ M BPB, the reversal by AA ranged from 54- 100%. These observations are compatible with the hypothesis that the exogenous AA is able to bypass the metabolic block induced by BPB, allowing synthesis of the AA metabolite(s) which is involved in the secretory event. DISCUSSION

These data suggest that PLA, activation is one of the common pathways involved in basophil histamine secretion induced by different stimuli. This is by no means a novel concept since several investigators have demonstrated that immune TABLE THE

EFFECT

OF

AA

ON INHIBITION

3

OF HISTAMINE

RELEASE

BY

ETYA

ETYA (pglml) Experiment

0

3

5

10

1 Antigen alone” Antigen + AA”

63” 67

60 35

37 10

10 1

2 Antigen alone’ Antigen + AA*

37 43

38 8

18 4

5 5

(I AgE. 5 x lO-3 &ml. b AA, 3 x 1O-6 M. ’ AgE, 1 x 1OF &ml. ” Percent histamine release.

238

MARONE,

KAGEY-SOBOTKA,

AND

TABLE EFFECI

OF AA

ON INHIBITION

4

OF HISI

Percentage Expt [AAl

0

I~ICH1ENS7EiN

1

.+RIINF R~I-r.-isr inhibition Expt

IO-” ,411

BI BPB

of HR”

2

Expt

3

0

IO ” ,I4

0

I 0 Ii .Lf

BPB.

I x 10 Ii M

x9

67

100

79

Xl

BPB, BPB,

3 s 10 i A4 I x IO-’ 41

47 ‘0

13 0

x7 71

Jh 71

i-.’ 36

7s.. 43 II

‘I Control histamine release (HR) to Rye grass Group I without and with IO ” M AA was ( I) 35 and 45%: (2) 26 and 28’?r: (3) 31 and 46%. Inhibition by BPB in the presence of AA wab calculated using the AA control.

activation of purified rat mast cells (8) and rat basophil leukemia cells (9) leads to the production of arachidonate, presumably as a result of the activity of PLA,. The hypothesis is supported by the clear-cut observations that two different drugs, p-bromophenacyl bromide and mepacrine, well-described inhibitors of PLA, (19-24), also block histamine release. Previous studies have demonstrated that BPB is relatively specific in its ability to block both PLA, activity (20) and the generation of arachidonic acid (25). Several indirect lines of evidence suggest that the mechanism by which BPB inhibits histamine release is related to PLAZ inhibition. First, the irreversibility of its effect is concordant with previous in \‘itt’o enzymatic studies (26). Second, the rapidity of the effect of BPB is likewise in accord with previous kinetic studies (21), indicating that the inactivation of PLA,, by BPB is extremely fast. Third, it is known that PLA, has an absolute catalytic requirement for Ca”+ and the Ca’+ binds to the enzyme but not at the active site (26, 28). After the enzyme binds calcium, it undergoes a conformational change (28) prior to phopholipid binding. In cell-free systems, CaS’ protects the purified enzyme against inactivation by BPB (28). Our results indicate that Ca’-~ impairs the inhibition of histamine release by BPB in a dose-dependent fashion. Analyses by Schild plot also suggest that CaS+ and BPB do not bind to identical sites on the enzyme. an observation which has received extensive experimental support ( 19. 28). The data derived from these studies are part of the increasing evidence that AA metabolism, via unidentified lipoxygenase pathways. is an essential element of the release of inflammatory mediators from human basophils stimulated either by IgE crosslinking or by other cell surface activation events. The partial reversal of BPB inhibition by exogenous arachidonate also suggests that PLA, activation triggers the AA cascade. This conclusion is. however, tempered by the assumptions regarding the specificity of BPB. While there is a large body of evidence in favor of this assumption of specificity it must, in the absence of actual measurements, remain just that. Further there is evidence, in rat mast cells, that AA may be generated by a diglyceride lipase (30). With these caveats aside, the available data tit the following hypothesis: the activation of specific cell surface receptors or the intracellular translocation calcium (by the calcium

PHOSPHOLIPASE

A,

AND

BASOPHIL

HISTAMINE

RELE.ASE

239

ionophore) activate basophil PLAz which catalyzes the hydrolysis of AA from cell membrane phospholipids. AA serves as a substrate for enzymes of the lipoxygenase pathway(s) which generate a product or products which are necessary for the release process. This hypothesis is based upon our own evidence (6, 7) and that obtained by others using rodent mast cells (22) or basophils (8). While our evidence is primarily pharmacologic and will need to be confirmed by direct biochemical measurements, these studies identify and partially characterize an important step in mediator secretion. REFERENCES 1. Lichtenstein, L. M., Marone, G., Thomas, L. L., and Malveaux, F. J., J. Invest. Dermutol. 71, 65. 1978. 2. Lichtenstein, L. M., Sohotka. A. K., Malveaux, F. J., and Gillespie, E.. InF. Arch. Allergy Appl. Immwwl. 56, 473, 1978. 3. Bourne, H. R., Lichtenstein, L. M.. Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer, G. M., Science 184, 19, 1974. 4. Plaut, M., Marone, G., Thomas, L. L., and Lichtenstein, L. M., “Advances in Cyclic Nucleotide Research” (P. Hamet and H. Sands, Eds.). pp. 161- 172, Raven Press, New York, 1980. 5. Flower, R. J., Pharmacol. Rev. 26, 33, 1974. 6. Marone, G., Sobotka, A. K., and Lichtenstein, L. M., J. Immunol. 123, 1669, 1979. 7. Marone, G.. Hammarstrom, S., and Lichtenstein. L. M.. C/in. Immunol. Immunopathol. 17, 117, 1980. 8. Kennerly, D. A., Sullivan. T. J., and Parker, C. W., J. Immunol. 122, 152, 1979. 9. Crews, F. T., Morita, Hirata, F., Axelrod, J., and Siraganian, R. P., Biochem. Biophys. Res. Commun. 93, 42. 1980. 10. Hogberg, B., and ijnvas. B., Acta Physiol. Scund. 41, 345, 1957. 11. King. T. P., and Norman, P. S., Biochemistry 1, 709, 1962. 12. Marsh, D. G., Milner, F. H., and Johnson, P., Inr. Arch. Allergy Appl. Immunol. 29, 521, 1966. 13. Ishizaka, I.. and Ishizaka, T., J. Immunol. 100, 554, 1968. 14. Hook, W. A., Schiffmann, E., Aswanikumar, S.. and Siraganian, R. P., J. Imnnmol. 117, 594. 1976. 15. Lichtenstein, L. M., J. Immunol. 114, 1692. 1975. 16. Marone, G., Plaut, M., and Lichtenstein, L. M., J. Zmmunol. 121, 2153, 1978. 17. Siraganian. R. P., Anal. Biochem. 57, 383. 1974. 18. Siraganian, R. P.. J. Immunol. Methods 7, 283. 1975. 19. Drenth, J.. Enzing, C. M., Kalk, K. H., and Vessies, J. C. A., Nature (London) 264, 373. 1976. 20. Volwerk, J. J., Pieterson, W. A., and de Haas, G. H., Biochemistry 13, 1446, 1974. 21. Roberts. M. F., Deems. R. A., Mincey, T. C.. and Dennis, E. A., J. Biochem. Chrm. 252, 2405, 1977. 22. Hirata, F., Corcoran. B. A., Venkatasubramanian, K., Schiffmann. E.. and Axelrod, J.. Proc. Nat. Acad. Sci. USA 76, 2640, 1979. 23. Flower, R. J.. and Blackwell, G. L., Biochem. Pharmacol. 25, 285, 1976. 24. Yorio, T., and Bentley, P. J., Nature (London) 271, 79, 1978. 25. Vogt. W., “Advances in Prostaglandin and Thromboxane Research” (C. Gallier al.. Eds.), p. 89, Raven Press, New York, 1978. 26. Slotboom, A. J., Jansen, E. H. J. M.. Vlijm, H., Pattus. F., Soares de Araujo, P.. and de Haas, G. H., Amer. Chem. Sot. 17, 4593, 1978. 27. Lichtenstein, L. M., and Osler, A. G., J. Exp. Med. 120, 507, 1964. 28. Pieterson. W. A., Volwerk, J. J., and de Haas, G. H., Biochemisrry 13, 1439, 1974. 29. Arunlakshana, 0.. and Schild, H. 0.. Brit. J. Pharmacol. 14, 48, 1959. 30. Kennerly. D., Parker, C., and Sullivan, T.. Fed. Proc. 38, 1018, 1979.