Arachidonic acid metabolic pathway of the rabbit placenta

PROSTAGLANDINS

ARACHIOONIC ACID METABOLIC PATHWAYOF THE RABBIT PLACENTA Michele H. Bloch, Laura L. McLaughlin, J. Crowley P. Needleman and A.R. Morrison

Departments of Pharmacology and Medicine Washington University School of r~dicine 660 South Euclid St. Louis, MO. 63110 ABSTRACT

Placenta ~crosomes prepared from animals late in gestation (29 days) e f f i c i e n t l y metabolize arachidonic acid into PG~, PGF2:, PG~, Tx~ and l i t t l e or no prostacyclin. In contrast to the late gestation placenta, the early (17 day) placental microsomes synthesize primarily PG~. The cytosolic (100,000 x g supernatant) fraction from early or late gestation placentae converted arachidonic acid, with a calcium dependent enzyme, into non-polar metabolites whose synthesis was inhibited by ETYA but not indomethacin. These metabolites were purified by HPLC and GC-MS analysis indicated the presence of 12-hydroxy-, ib-hydroxy-, and 11-hydroxy-eicosatetraenoic acid. The ~ t o chondrial (8,000 x g pellet) produced PG~; PGF2~; 12-, 11-, 15-HETE; the C-17 fragment HHT; and the unusual cyclooxygenase metabolite 15-keto-PG~. These biologically active metabolites may play a v i t a l role in the reproductive function of the placenta. INTRODUCTION

The placenta has a unique and vital role during fetal development. It transports nutrients from the maternal to the fetal c i r c u l a t i o n , and through as yet uncharacterized mechanisms, is thought to regulate fetal growth ( I ) , to produce the large amounts of steroids required during pregnancy (2). The placental trophoblast synthesizes several glycoprotein hormones, of which the best known are placental lactogen and chorionic gonadotropin (3). Addit i o n a l l y , placental factors may play a role in maintaining the immunologically protected state of the fetus (4). However, our understanding of placental function is very rudimentary. The placenta and fetal membranes contain considerable amounts of arachidonic acid, and metabolites of arachidonic acid are of c r i t i c a l importance in reproductive processes (5). We have studied the metabolism of arachidonic acid by the placenta of a species suitable for experimental study: the rabbit. We report herein that this tissue metabolizes arachidonic acid to several mono and dihydroxy derivatives, which we have i d e n t i f i e d and characterized. In addition, we describe the a b i l i t y of the placenta to metabolize arachidonic acid by the cyclooxygenase pathway. Moreover, we have compared the a b i l i t y of early and late placentae to produce prostaglandins as a step towards understanding the biological function of these compounds. MATERIALS AND METHODS

Time pregnant rabbits (27-29 days pregnant) were from Boswell Bunny Farm (Pacific, Missouri). [ 1_14 C]-arachidonic acid (55 mCi/mmol) and 5,6,8,9,11,12,13,1b 3H) arachidonic acid (4b.b Ci/mmel) were purchased from New England

FEBRUARY 1985 VOL. 29 NO. 2

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Nuclear. Arachidonic acid was from Nu-Chek. B i s ( t r i m e t h y l s i l y l ) t r i f l u o r o acetamide (8STFA) with i% trimethylchlorosilane was from Pierce Chemical Co. Activated s i l i c i c acid (Unisil) was from Clarkson Chemical Co. HPLC grade solvents were purchased from Mallinckrodt Chemical Co. All other chemicals were analytical reagent grade. Thin layer chromatography was performed on Polygram Sil-G plates from Brinkman Instruments, Inc. Kodak XAR-5 film was used for autoradiography. Preparation of placental 8,000 x 9 pellet, i00,000 x ~ pellet and 100,000 x 9 supernatant. Pregnant animals were purchased either at middle (ib-18 days) or late (2b-28 days) pregnancy; gestation in rabbits is 31 days. As placental development occurs during the later 2/3 of the gestation, early placentae were obtained from animals in middle pregnancy, and late placentae from animals in late pregnancy. The animal was anesthetized with pentobarbital (30 mg/Kg), the abdomen incised, and the gravid uterus removed and placed in ice cold 50 mM Tris buffer, pH 7.2, containing 2 told EGTA. Subsequently, the tissue was kept at 0-4%. The uterine horns were opened and the placentae dissected away from the uterus and fetal tissues. Isolated placentae were blotted, weighed, and placed in 3 volumes of fresh buffer. The tissue was minced, homogenized, using a Tekmar tissumizer, and centrifuged at 8,000 x g for 15 minutes. The supernantant was decanted through a layer of cheesecloth and centrifuged at 100,000 x g for one hour. The 100,000 x g supernatant was decanted and reserved, and the microsomal pellet resuspended in buffer using a volume equal to 1/4 the original tissue weight. Protein was measured by technique of Lowry (6). Microsome incubations for thin la~er chromatography. The microsomal preparation (100 ~ l ) was incubated witn LI"C] arachidonic acid (18 ~M; 300,000 cpm, or as indicated) or with [14C] prostaglandin endoperoxide PGH2 (8.7 pM; 140,000 cpm) at 37°C in a total volume of 150 microliters. Incubations of both early and late placentae contained between I and 3 mg protein. 1.2 mM L-epinephrine, 1.0 mM glutatnione, 5 pg/ml indomethacin, 5 mM imidazole or 25 ~g/ml ETYA were added to the incubation mixtures as described. The reaction mixtures were incubated at 37°C for the indicated times, acidified to pH 3.0 with i N HCI and extracted three times with 2 volumes of ethyl acetate. The ethyl acetate layer was dried with a stream of nitrogen, and resuspended in a small volume of chloroform/methanol ( 2 / I ) . Fhe material was subjected to thin Iayer chromatography i n system A-9 (the or gani c phase of ethyl acetate:water:iso-octane:acetic acid, II0:I00:50:2U) which permits the separation of 6KPGFIc (7) or in system C (chloroform: methanol:acetic acid:water 90:8:l:O.~) which permits the separation of thromboxane 82 (TxB2) from other prostaglandins (8). Occasionally, TxB2 can be separated from PGE2 in system A-9, as seen in Fig. i . Unlabelled prostaglandin standards were added to the concentrated extracts before plating and were visualized with iodine vapor; the radioactive zones were detected by autoradiography. Incubations of 100,000 x ~ supernatant f o r t h i n l a y e r chromatography. IO0,O0O x g supernatant ( i 0 0 ~ I ) was incubated at- 37 ° w i t h [ 1 " C ] - a r a c h i d o n i c acid in a t o t a l volume of i b 0 ~ l ; i n c u b a t i o n s of both e a r l y and l a t e placentae contained between 1.5 and 2.5 mg t o t a l p r o t e i n ; 5 mM CaCI2, 5 ~g/ml i n d o methacin or 5 pg/ml ETYA were added, as i n d i c a t e d . The r e a c t i o n was t e r m i nated by the a d d i t i o n of 3 volumes of methanol. The m i x t u r e was c e n t r i f u g e d and the p e l l e t r e e x t r a c t e d w i t h i volume of methanol. The combined m e t h a n o l / w a t e r e x t r a c t s were d r i e d over a stream of n i t r o g e n and chromato-

204

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METABOLISM OF [14C]-AA OR [14G]-PGH , BY

RABBIT PLACENTAL MIGROSO&~S: SYSTEM A-9 MICROSOMES* ~4C]-AA

EPI

MICROS(~IES • [14cl- pGH2

EPI

+

PGH,

GSH

ALONE

®

O m

m i ~

Fig.

i.

0

Z

Placental microsomes prepared from animals late in gestation were

incubated with e i t h e r prostaglandin utes.

ORIGIN

[IwC] aracnidonic acid (18 pM, 300,000 cpm) or [14C]

endoperoxide PGH~ (8.7 pM, 140,000 cpm) at 37°C for

30 min-

L-epinephrine (1.2 mM) and GSH ( i mM) were added to incubations with

[14C] arachidonic acid as indicated. graphed in

Extracts of incubations were chromato-

system A-9 and autoradiographed.

Column 3 depicts

microsomes

incubated with [14C] prostaglandin endoperoxide PGPe and column 4 depicts the incubation of PGHe in buffer without microsomes. The positions of unlabelled prostaglandin standards are indicated in the margin.

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PROSTAGLANDINS

graphed in system BDEA (benzene:diethyl ether:ethanol:acetic acid 50:40:2: 0.2) (9). The radioactive zones were visualized by autoradiography and, where indicated, the radioactive zones were cut out from the plate, and counted in 4A20 liquid s c i n t i l l a t i o n counting solution (Beckman). Incubations of 8,000 x ~ pellet for thin layer chromatography The 8,000 x g pellet was resuspended in an equal volume (w/v) of buffer equivalent to the original wet weight of placenta (protein concentration 7-12 mg/ml). Incubations were performed at 37% for 10 min and contained enzyme suspension, co-factors and [1-~ ~] arachidonic acid (17 pM) in a total volume of 150 p l . The reaction was terminated with 4 vols MeOH. The MeOH fraction was evaporated under ~ and subjected to s i l i c a gel thin layer chromatography. Isolation of products of HPLC. Preparative isolation for GC-MS analysis was carried out on products generated by incubation of fatty acid with 8,000 x g p e l l e t , 100,000 x g supernatant and 100,000 x g p e l l e t . C o l d arachidonic acid was used with [l~C]-arachidonic acid tracer in multiple small incubations performed at 37°C for 30 minutes, and stopped by the addition of 3 volumes of methanol. The ~ x t u r e was centrifuged and the pellet re-extracted with one volume of methanol. The combined methanol/water extracts were acidified to pH 3.b with 0.I N HCI, and extracted with four volumes of diethy| ether and two volumes of water; the diethyl ether layer was then washed with 2 volumes of water. Ethanol was added to the ether layer to form an azeotrope with water and the ether layer was evaporated under reduced pressure. The reaction ~ x t u r e was chromatographed on an open bed s i l i c i c acid column (I gram). The ether extract was evaporated to dryness, applied to the column and eluted successively with 30 ml hexane/ether (92/8), 30 ml hexane/ether (55/45), 30 ml ethyl acetate, and 30 ml methanol. The various fractions were concentrated and reserved for HPLC separation. HPLC analysis. HPLCanalysis u t i l i z e d a pump (Model 6000A) and an inject o r (Model U6K) purchased from Waters Instruments. Compoundswere detected both spectrophotometrically, with a Model 450 variable wave-length spectrophotometer, from Waters and by monitoring radioactivity, by liquid s c i n t i l l a t i o n counting. Preparation of trimethyl s i l y l ether methyl ester derivatives for 9as chromatography-mass spectrometry. Excess diazomethane in diethyl ether was added to HPLC purified samples dissolved in 50 ~I methanol. The reaction was carried out for 10 minutes at room temperature. The ether was then evaporated with a stream of nitrogen, and a few drops of methanol added and dried again with a stream of nitrogen. The residue was dissolved in 10 pl pyridine and IU ~1 of bis(trimethylsilyl)tri-fluoroacetamide (BSTFA) added. The reaction was incubated overnight at room temperature. Gas Chromatography-Mass Spectrometry. Mass spectrometry of the methyl ester t r i m e t h y l s i l y l ethers was performed on a HP5985B system at the Washington University Resource Center. The column was a 25 meter cross linked OV-1 wide bore capillary column and programmed from 85% to 240°C at 30°/min. with helium as carrier gas. The injector was a Grob type and was set at 250%. C values were obtained on an HP 5830-A gas chromatograph, on a I meter x 2 mm I.D. column, packed with 3% OV-1, run isothermally at 220%, with carrier gas N~.

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PROSTAGLANDINS

RESULTS The extracts of incubations of ~crosomes prepared from late placentae were chromatographed in system A-9 (Fig. 1). Late placental microsomes e f f i ciently metabolized arachidonic acid to PG~, PGF2:, and l i t t l e or no 6KPGFI:. In addition, several unidentified non-polar compounds were produced. Altering the concentration of arachidonic acid between 3 and 80 ~M did not raise the concentration of 6KPGFIa produced (not shown). A similar spectrum of products was observed when late placental microsomes were incubated with prostaglandin endoperoxide PG~ (Fig. l - t h i r d column). The microsomes synthesized PG~=, PG~. PG~, but only trace amounts of 6KPGFI~. Column 4 shows the breakdown of PG~ to PG~ and PG~ in buffer without microsomes. The addition of ~crosomes to PG~ diminishes the non-enzymatic formation of PG~, and f a c i l i t a t e s the PG~= but does not result in prostacyclin formation. As PG~ and Tx~ comlgrate in system A-g, the identity of the product in this region iS in doubt; therefore, we chromatographed placental' incubations in system-C, which permits resolution of TxB2 (Fig. 2). Late p l a c e n t a l microsomes also synthesized a material w h i c h comigrated with authentic T x ~ , whose formation from either arachidonic acid (column i) or prostaglandin endoperoxide PG~ (column 4), was inhibited by the thromboxane syntnetase i n h i b i t o r imidazole (columns 3 and 5). This is strong evidence for the production of Tx~ by this tissue. We next determined the spectrum of arachidonic acid metabolites produc@d by microsomes from early gestation placentae. In contrast to the late gestation placenta, the early tissue synthesized primarily PGE2 and only trace amounts of Tx~, PG~ and PG~ as shown by chromatography is system C (Fig. 3). Whensimilar extracts from early tissue were chromatographed in system A9, no 6KPGFI: production was observed (not shown). The IO0,OUO x g supernatant of rabbit placenta was incubated witn [zwC]arachidonic acid and the extracts analyzed by thin layer chromatography. In the presence of Ca2+, arachidonic acid was converted to a number of non polar compounds whose synthesis was inhibited by ETYA but not by indomethacin (not shown). To generate s u f f i c i e n t material for i d e n t i f i c a t i o n , large scale incubations were performed as described. Open bed s i l i c i c acid chromatography of the ether extract of the large scale incubation mixtures was useful both to accomplish a preliminary separation of the compounds, and to remove many biological impurities. The hexane/ether 55/45 fraction, which contained primarily mono-hydroxy eicosanoids, was subjected to s i l i c i c acid HPLC (Fig. 4). Three major peaks of radioactivity were detected at retention volumes of 10 ml (1) 18 ml ( I I ) and 22 ml ( I l l ) each of which coincided with a peak of u l t r a v i o l e t absorption at 235 nm. In addition, two minor peaks of radioactivity were detected at 38 ml (IV) and 45 ml (V) which also absorbed at 235 nm. Compounds I I and I l l chromatographed at the same position as authentic standards of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 15-hydroxy-b,8,11,13-eicosatetraenoic acid (15-HETE) run on this column, respectively. Compounds I and V were unidentified but compounds I f , I l l , and IV proved to be 12-hydroxy, 15-hydroxy and 11-hydroxy eicosatetraenoic acid respectively based on GC-mass spectrometric analysis (Table i ) . In addition, the lO0,OOO x g supernatant produced small amounts of 5,6 dihydroxy-8,11,14 e i c o s a t r i e n o i c acid (C value 22.b) with ions at 496 (M.+), 406 (M-90), 30b clevage between carbons 6 and 7, ZI5 (30b-90), 203 clevage between carbons 5

FEBRUARY 1985 VOL. 29 NO. 2

207

PROSTAGLANDINS Table i .

Compounds I I ,

I I I and IV were subjected to mass spectral analysis as

described in Materials and Methods.

The ion peaks and C-values obtained are

shown.

Compound

II

C-value

21.2

(12-HEFE)

ion peaks m/z

406 (M), 391 (M-15), 375 (M-31), 295 ( M - I l l loss of CH2-CH=CH-(CH2) CH3, 229, 205 (295-90), 173, 145, 131.

IiI

21.3

316 (M-90), 225 (M-188).

(15-HETE)

IV (II-HErE)

208

406 (M), 391 (M-15), 335 (M-71),

21,5

225 (M-181) base peak 406 (M), 391 (M-15), 375 (M-31).

FEBRUARY 1985 VOL. 29 NO. 2

PROSTAGLANDINS

o,

o.

BY RABBIT PLACENTAL MICROSOMES: SYSTEM C i

MICROSOMES+ ['4C]-AA EPI

EPI +

MICROSOMES+[~tC]-PGHI

EPI +

+

GSH IMID

W

Fig.

ZL.

Placental

P(;H=

IMID ALONE

m

D

microsomes prepared from animals late in gestation were

incubated with either [14C] aracnidonic acid (18 I~M, 300,000 cpm columns I-3) or [I4C] prostaglandin endoperoxide PGH2 (8.7 ~M, 140,000 cpm columns 4-6) at 37°C for

30 minutes;

1.2 mM L-epinephrine,

imidazole

were added as indicated.

chromatographed in system C. [14C]

prostaglandin

1.0 mM glutathione,

Extracts

of

reaction

or 5 mM

mixtures

were

Column 6 depicts the spontaneous breakdown of

endoperoxide PGHe in

positions of unlabelled prostaglandin

FEBRUARY 1985 VOL. 29 NO. 2

buffer

without microsomes.

The

standards are indicated in the margin.

209

PROSTAGLANDINS

r

1

[14CJAA METABOLISM BY MICROSOMES OF EARLY AND LATE GESTATION PLACENTA LATE 29DAY

Fi~. 3.

EARLY 29DAY

. . . . . . .

~^v

Microsomes prepared from late or early placentae were ncubated with

~4C] aracbidonic acid (36 ~M, 600,000 cpm) in the presence of 1.2 mM L-epinephrine at 37°C for b minutes. autoradiographed;

the

position

indicated in the margin.

Extracts were cnromatographed in system C and of

unlabelled prostaglandin standards are

Extracts of incubations of 2 late and 2 early pla-

centae are shown.

210

F E B R U A R Y 1985 VOL. 29 NO. 2

PROSTAGLANDINS

0.4

:3. LO C'J

E

E ~3 r~3 (M

I

© ;,,(

1"

c5 O

E

I

13..

--- I 0

F.i 9.

4.

HPLC t r a c i n g

phase s i l i c i c flow

I 16

8

I

I

[

l

I

56 24 32 4 0 48 VOLUME ELUTED ( m l )

of

hexane/e~her

(55/45)

I

64

f2

fraction

separated on normal

acid using a mobile phase of h e x a e : e t h a n o l : a c e t i c acid 994:6:1

r a t e t m}/min.

Peaks I I

through

IV are described in Table I.

Peaks I

and V here u n i d e n t i f i e d .

F E B R U A R Y 1985 VOL. 29 NO. 2

211

PROSTAGLANDINS

and 6 and similar to spectrum described by Oliw et al. (i0) Incubation of 8,000 x g pellet with arachidonic acid followed by organic extraction and HPLC separation provided several products (Fig. 5) which were i d e n t i f i e d by GCMS table I f . The compound labelled E is 15 oxo PGE2 an unusual metabolite of arachidonic acid produced in the absence of pyridine nucleotides and f u l l y described in another paper ( I I ) . DISCUSSION We have demonstrated that both early and late gestational agent placenta metabolized aracnidonic acid by the cyclooxygenase and lipoxygenase pathways. The late placenta synthesized PGF2~, [xB2, PG~, PG~, and l i t t l e or no 6KPGFI:, while the placentae f r o m earlier animals synthesized primarily PG~. In the presence of Ca~ both early and late placentae synthesized monoand dinydroxy eicosanoids. By late in gestation the placenta has acquired the a b i l i t y to synthesize PG~, P G ~ , Fx~, and trace amounts of 6KPGFI:. Presumably, the function of the l a t t e r compounds must be restricted to late pregnancy, as for example, at parturition.

The minimal capacity to produce prostacycIin by this vascular bed is s t r i k i n g because of the presumed importance of prostacyclin to prevent platelet aggregation and adhesion to blood vessel walls. In contrast to the placenta, other vascular beds produce primarily prostacyclin (12) as do cultured endothelial cells (13). O t h e r mechanisms must exist to prevent platelet aggregation and adhesion in the placental vascular bed. There is evidence in the l i t e r a t u r e to support this conclusion. Although microsomes of human placenta do not synthesize prostacyclin from either arachidonic acid or prostaglandin endoperoxide PG~ (14,15), the human placenta is reported to contain an i n h i b i t o r of platelet aggregation (14,16). A v a r i e t y of tissues are now known to possess ]ipoxygenase a c t i v i t y , i n c l u d i n g p l a t e l e t s , leukocytes, and the renal cortex [17-19]. 12-HETE is the primary product of the p l a t e l e t lipoxygenase. This compound has chemotactic a b i l i t y towards eosinophi|s, and polymorphonuclear ]eukocytes, and thus ~ y play a role in the inflammatory response [20]. 15-HEFE is produced by rabbit polymorpnonuclear leukocytes and is able t o i n h i b i t leukocyte biosynthesis of b-HETE and b,12-diHEfE [21]. The f i n d i n g that the placenta is a rich source of lipoxygenase enzymes suggests that the b i o l o g i c a l u t i l i z a t i o n of these compounds is more widespread than previously a n t i c i p a t e d . The placenta serves a number of c r i t i c a l functions during f e t a l development. The placental vasculature contacts maternal blood at the i n t e r v i l l o u s space permitting an exchange of materials by a counter-current exchange mechanism [22]. Transport of nutrients and oxygen from the maternal to f e t a l compartments and of waste products from f e t a l to maternal compartments occurs by a v a r i e t y of processes. The trophoblast is also the s i t e of synthesis of numerous steroid and peptide hormones [2]. A d d i t i o n a l l y , i t has often been suggested that placental factors may p a r t i c i p a t e in two other c r i t i c a l functions during pregnancy: the maintenance of the immunological protection of the fetus, and tne regulation of fe t a l growth [ 1 , 4 ] . The factors which regul a t e these placental functions are largely unknown. We suggest that the lipoxygenase d e r i v a t i v e s we have observed are candidates to p a r t i c i p a t e in these placental functions. Metabolism of arachidonic acid by the rabbit placenta is not l i m i t e d to the lipoxygenase pathway. We have recently shown

212

F E B R U A R Y 1985 VOL. 29 NO. 2

PROSTAGLANDINS

0 x

E

(3. 0 0

° 1

I

0

Fig. b

1

20

40 60 MINUTES

80

I00

HPLC tr a c i n g of organic extract of 8,000 x g p e l l e t incubated with

aracnidonic

acid

in

e t h a n o l : a c e t i c acid, gradient to rains.

I'

presence 994:6:b

of (A)

5 mM Ca2+. run

isocratic

80% hexane:ethanol:acetic acid

Flow rate 3 ml/min.

norma] phase s i l i c i c

acid.

Mobile for

phase

is

hexane

25 rains than a l i n e a r

(90:I0:0.b),

solvent B over 60

The column is a semipreprative ~ Porasil (Waters) PeaKs A through G are described in Table I I .

FEBRUARY 1985 VOL. 29 NO. 2

213

PROSTAGLANDINS Fable i I

Compound

C-value

ion peaks m/z.

A /2-HETE

21.2

406M+, 391(M-15), 375(i~-31) 295(M-III) loss of C~CH=CH (C~)4C~

8 15-HETE

21.3

406(M+) 391(M-15), 335(M-71) 316(M-90), 225(M-181)

C II-HEFE

21.5

225(M-18[) base peak 406(~+), 391(M-15), 375(M-31)

I) HHT

19.3

366(M+), 295(M-71), loss of (CH2), CH3, 225(M-141) loss of CII2CH=CH(CH2)3COOH

E /5-Keto

24.2,24.6

494(M+), 479(7-15), 463(7-31), 406(7-31), 404(7-90). 373(M-(90+31)), 321(M-73) ]oss of carbons 9,10,11,methoxine and OSi(CH3)3

F PGEa

24.2, 24.6

539(M+), 524(M-15), 508(M-31), 449(M-90), 461(M-71), 418 (M-(90+3)), 371(M-(90+7L)).

G PGF2m

23.9

584(~+), 513(M-71), 494(g-90), 490(M(90x2)).

214

F E B R U A R Y 1985 VOL. 29 NO. 2

PROSTAGLANDINS that tnis tissue e f f i c i e n t l y metabolizes arachidonic acid by the cyclooxygenase pathway as well. Fhe placenta is a complex organ, containing several c e l l types, which changes c o n t i n u a l l y over the course of gestation. Placental c e l l s derive from the t r o p n o b l a s t i c c e l l mess, the outer cell layer of the blastocyst. Init i a l l y , the placenta consists of an inner layer of mononuclear cytotrophoblast and an outer layer of multinucleated s y n c t i o t r o p h o b l a s t , which forms by fusion of cytotrophoblast c e i l s [23]. The placenta invades the uterine endometrium, engulfing meternal c a p i l l a r i e s , eventually forming a blood f i l l e d i n t e r v i l l o u s space. Mesenchymal c e l l s enter the placenta and, with the other cell types, c o n t r i b u t e to the formation of placental v i l l i . The connective tissue core of the placental v i l l i w i l l eventually contain f i b r o b l a s t s , Hofbauer c e l l s , and c a p i l l a r i e s [24]. Tnus, there are a large number of c e l l s which are candidates for the production of lipoxygenase products; c l e a r l y assignment of s y n t h e t i c capacity to one would aid in determining function. Additionally, we have recently found that two other tissues derived from the embryonic tropnoblast actively metabolize arachidonic acid. The rabbit amnion and yolksac splanchnopleure e f f i c i e n t l y metabolize arachidonic acid by both the cyclooxygenase and lipoxygenase products [25]. In conclusion, we nave shown that the rabbit placenta, a tissue of trophoblastic origin actively synthesizes a variety of lipoxygenase products proceeding from 15, 12, and 11 lipoxygenation. We suggest that these compounds may play a vital role in function of these tissues. ACKNOWLEDGEMENTS This work is supported by NIH part HL1439 and HL20787 (PN) and 30562-02 (ARM). Dr. ~orrison is an established i n v e s t i g a t o r of the American Heart Association.

FEBRUARY 1985 VOL. 29 NO. 2

215

PROSTAGLANDINS

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. g. I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. ZU.

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FOX, H. and Elston, C.W. (1978) in Pathology of the Placenta (Saunders, W.B ed.) Vo]. VII, pp. 38-49, Philadelphia, Pennsylvania. Pierce, G.B. and Midgley, A.R. (1963) Am. J. Path. 43, 153-173. Fox, H. and Elston, C.W. (1978) in Pathology of the Placenta (Saunders, W.B ed.) Vol. VII, pp. 1-37, Philadelphia, Pennsylvania. E l l i o t t , W.J., Bloch, M.H., McLaughlin, L.L., and Needleman, P. (1983) Fe Proc. 42, 349. Editor:

216

H a r o l d R. B e h r m a n

Received: 7-25-84 Accepted:ll-20-84

F E B R U A R Y 1985 VOL. 29 NO. 2