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BBA 51170

Biochimica et Bwph_ysrca Acta, 7 12 ( 1982) 408-4 I I Elsevier Biomedical Press

RADIOIMMUNOLOGIC IDENTIFICATION OF PROSTAGLANDINS PRODUCED BY SERUM-STIMULATED MOUSE EMBRYO PALATE MESENCHYME CELLS

IFTEKHAR ALAM a, ANNA M. CAPITANIO b, J. BRYAN SMITH a, KENNETH P. CHEPENIK ’ and ROBERT M. GREENE’

a Cardeza Foundation, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107 (U.S.A.), ’ Haemophilia and

Thrombosis Center Angelo Bianchi Bonomi, University of Milano, Milan0 (Italy) and ‘Daniel Baugh Institute of Anatomy, Thomos

Jefferson University, Philadelphia, PA I91 07 (U S.A.)

(Received November 17th. 1981)

(Revised manuscript received May 6th, 1982)

Key words: Prosraglandin; Arachidonate metabolism; Serum stimulation; (Mouse palate mesenchyme cell)

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F,, (the stable product of prostacyclin, prostaglandin I,), prostaglandin E, and prostaglandin Fza. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E, (36-fold), 6-ketoprostaglandin F,, (15-fold) and prostaglan- din Fza (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.

Introduction

Recently Greene et al. [l] reported that pros-

taglandin I, stimulates cyclic AMP synthesis in

mouse embryo palate mesenchyme cells. This sug-

gests that prostaglandins may play an as yet unde- fined regulatory role in the palate development

process. We therefore investigated the ability of

mesenchyme cells to synthesize prostaglandins. Our initial studies [2] described the qualitative nature of radioactively labeled prostaglandin-like com-

pounds released from mouse embryo palate mesenchyme cells prelabled with [ I4 Clarachidonic acid in response to several stimulating agents. Fur- ther, the increase in the radioactivity correspond- ing to the prostaglandins was inhibitable by the cyclooxygenase inhibitor, indomethacin. We now demonstrate the production of different endoge- nous prostaglandins by radioimmunoassay (RIA) after their purification by high-performance liquid chromatography.

Materials and Methods

A/J or C57BL6J mice were killed on the 14th day of pregnancy and embryonic palatal and max-

illary processes dissected, pooled, minced and dis- sociated with 0.25% trypsin/O. 1% EDTA in Ca2+ -

and Mg’-free phosphate-buffered saline for 10

min at 37°C to yield a single suspension which

was seeded into 35-mm tissue culture dishes

(Falcon) at a density of 1.25 . lo5 cell/cm2.

Cells were grown for 24 h at 37°C in humidified

air containing 5% CO,, washed with fresh media, and then incubated for 10 h in 1 ml of fresh media

which was 10% in fetal calf serum (complete media). In some cases the complete media was 10

PM in 5,&l 1,lCeicosatetraynoic acid. Cell culture fluid (5 ml) was acidified to pH 3.5

with formic acid. Small XAD-2 columns (Isolab, Akron, OH) were prepared by washing with 20-25 ml of distilled water. The acidified fluid was passed through the XAD-2 column, and the effluent re-

OOOS-2760/82/0000-0000/$02.75 0 1982 Elsevier Biomedical Press

409

chromatographed on the same column again. The

column was then washed with 20-25 ml of 100% ethanol. The ethanol was evaporated in a vacuum

concentrator (Savant Co., Hicksville, NY). The

residue was reconstituted in acetonitrile/ethanol

(1: 5, v/v) to a final volume of 80-100 ~1 for

fractionation by high-performance liquid chro-

matography (HPLC). For HPLC, we used a Waters Liquid Chro-

matograph (Milford, MA) equipped with two

model 6000A Solvent Delivery Systems. The col-

umn employed was a 30 cm X 3.9 mm fatty acid

analysis column used in the reversed-phase mode.

The arachidonic acid metabolites were eluted iso- cratically with a solvent system containing

acetonitrile/benzene/acetic acid/water (230 : 2 : 1 : 767). The flow rate was 2.0 ml/mm per tube. 60 tubes were collected for each run. Each fraction

was evaporated to dryness by vacuum concentra-

tion and resuspended in 0.5 ml of NaC1/0.14 M

Tris-HCl/O.Ol M buffer, pH 7.4, prior to RIA.

The fractions were analyzed for 6-ketoprostaglan-

din F,,, thromboxane B,, prostaglandin Fz, and

prostaglandin E, using antibodies prepared against

each of these prostaglandins at the Cardeza

Foundation.

All solvents were glass-distilled and water was

purified and degassed by filteration through a

Millipore filter (0.45 pm) under vacuum with stir-

ring. Authentic prostaglandins were a generous gift from Dr. John Pike, The Upjohn Company,

Kalamazoo, MI and 5,8,11, lbeicosatetraynoic acid

was a gift from Dr. W. Scott, (Hoffman-LaRoche, Nutley, NJ).

Results

The chromatographic profile of arachidonic acid metabolites from serum-treated mesenchyme cells

was compared with the profile of standard pros-

taglandins (Fig. 1). The retention properties of

standard prostaglandins are shown in panel A, the

total amounts of each metabolite found in serum- treated mesenchyme cells in panel B, and the control serum-treated media are shown in panel C. Serum stimulated the synthesis of prostaglandin E, by 36-fold, 6-ketoprostaglandin F,, 15-fold and prostaglandin FZol by 7-fold above control values. By contrast, there was little change in the values

CELLS + I .2 COMPLETE

MEDIA

0.6

0.4

0.2

0

RET

IO 20 30 40

‘ENT ION TIME _ (min)

Fig. 1. The elution patterns of four cyclooxygenase metabohtes

of arachidonic acid separated by HPLC and analyzed serologi-

tally as described in Materials and Methods. Panel A shows the

profiles for standard metabohtes. Panel B gives the profile of

stimulated prostaglandins by mouse embryo mesenchyme cells

in complete media and panel C gives the patterns of complete

media only. K-PG, ketoprostaglandin; PG, prostaglandin; TX,

thromboxane.

for thromboxane B, between control serum-con-

taining media (0.57 ng/ml) and the serum-treated cells (0.66 ng/ml). High control values of throm- boxane B, are not unexpected as the serum present in experimental media is likely to contain this

product as a result of platelet activation. Inhibition of prostaglandin synthesis. The en-

zyme cyclooxygenase which initiates prostaglandin synthesis can be inhibited by 5,8,1 l,lCeico-

410

TABLE I

INHIBITION OF PROSTAGLANDIN SYNTHESIS BY 5,8,1 I,l4-EICOSATETRAYNOIC ACID

Data is expressed in pmol/ml. For cells in complete media, values are mean2S.D. (four determinations). For 5,8,11,14-eicosa-

tetraynoic acid cells, cells were treated with complete media which was 10 pM in 5,8,11,14-eicosatetraynoic acid for 2 h, washed twice

with media only, and then incubated for 10 h with complete media.

Prostaglandin E, Prostaglandin Fla 6-Ketoprostaglandin F,, Thromboxane B,

Cells in complete media 3.56-cO.55 0.96 to.03 1.02*0.35 1.78-cO.15

5,8,11,14-Eicosatetraynoic acid

cells in complete media 0.30 0.26 0.20 1.79

Complete media only 0.21 0.16 0.13 1.56

satetraynoic acid and indomethacin [3,4]. Table I

illustrates the effect of 5,8,11,14-eicosatetraynoic

acid on serum-stimulated prostaglandin levels.

Pretreatment of mesenchyme cells for 2 h with 5,8,11,14-eicosatetraynoic acid ( 10 PM) before

challenging with serum markedly inhibited the in-

creases in prostaglandins and the values were close

to the basal levels.

Discussion

The separation of the cyclooxygenase products

of arachidonic acid using HPLC and their mea-

surement by RIA have been described previously [5] and reviewed in detail elsewhere [6]. In brief,

the cyclooxygenase products, 6-ketoprostaglandin

F,,, thromboxane B,, prostaglandin FZa and pros- taglandin E, are resolved on a fatty acid analysis

column under isocratic conditions and measured

using antibodies raised against the individual pros-

taglandin. Identification of each prostaglandin is

based on its retention time, determined using a

known standard, and its serological specificity. The present results show that mouse mesenchyme

cells synthesize prostaglandin E,, 6-ketopros-

taglandin F,, and prostaglandin FZa when stimu- lated by serum and that this synthesis is inhibited by 5,8,ll,lCeicosatetraynoic acid.

Different cell types may produce prostaglandins in different relative proportions in response to various stimulating agents [7]. The relative amounts of prostaglandins synthesized by embryonic palatal mesenchyme cells in response to serum consisted of 60% prostaglandin E,, 25% 6-ketoprostaglandin F,, and 12% prostaglandin F,,. Similar relative

proportions of these prostaglandins have been ob-

served to be biosynthesized by other cell types in

response to human or bovine serum [7]. The mechanism of the stimulation of pros-

taglandin synthesis by serum is not clear. It has

been reported that serum contains phospholipase A, activity which may release arachidonic acid

from the cell membrane [8,9]. Alternatively, the

stimulation could be due to platelet-derived growth

factor, a polypeptide released from platelet alpha

granules during blood clotting. It was recently

proposed that the presence of this polypeptide in

serum was the explanation for activation of pros- taglandin synthesis in Swiss mouse 3T3 fibroblasts

[lOI. While it was observed in a previous study that

mouse embryo mesenchyme cells produce radioac-

tive prostaglandins from radioactive arachidonic

acid [2], it was important to determine the identi-

ties and relative amounts of the prostaglandins

produced endogenously by these cells as we have done here. Future studies will be directed to de-

termining what role, if any, these prostaglandins play in palate development.

Acknowledgements

This work was supported in part by NIH grants HL-14890 and DE-05550. R.M. Greene is a recipi- ent of NIH Research Career Development Award K04-DE00095. A. Capitanio was supported in part by Fondazione Floriani, Milan, Italy.

411

References

1 Greene, R.M., Lloyd, M.R. and Nicolaou, K.C. (1981) J.

Craniofacial Genet. Devel. Biol. 1, 261-272

2 Chepenik, K.P. and Greene, R.M. (1981) B&him. Biophys.

Res. Comm. 100, 951-958

3 Hamberg, M. and Samuelsson, B. (1974) Proc. Natl. Acad.

Sci. U.S.A. 71, 3400-3404

4 Ahern, D.G. and Downing, D.T. (1970) Biochim. Biophys.

Acta 210, 456-461

5 Alam, I., Ohuchi, K. and Levine, L. (1979) Anal. Biochem.

93, 339-345

6 Alam, I. and Levine, L. (1981) Methods Enzymol. 73,

275-288

7 Levine, L. (1979) in Hormones and Cell Cultures (Sato.

G.-H. and Ross, R., eds.), Book, B. pp. 632-640, Cold

Spring Harbor Laboratory, Cold Spring Harbor

8 Kaplan-Harris, L., Weiss, J., Mooney, D., Beckerdite-

Quagliata, S. and Elbasch, P. (1980) J. Lipid Res. 21,

617-624

9 Van den Bosch, H. (1974) Annu. Rev. B&hem. 43,243-277

10 Shier, W.T. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 137-141