Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Vol. 110, No. 1, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 14, 1983

Pages 250-255

SYNTHESIS OF PROSTAGLANDIN E2 AND PROSTAGLANDIM F2a BY TOADFISH RED BLOOD CELLS Lauren M. Cagen, Zahir Qureshi, and Hiroko Nishimura Departments of Pharmacology and Physiology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163, and Division of Science and Mathematics, Rust College, Holly Springs, Mississippi 38635 Received November 15, 1982

SUMMARY: S a l i n e washed red blood c e l l s of the t o a d f i s h convert [ I - t 4 C ] a r a c h i d o n i c acid to products t h a t cochromatograph w i t h p r o s t a g l a n d i n E 2 and p r o s t a g l a n d i n F2a. This s y n t h e s i s i s i n h i b i t e d by indomethacin (10 ug/ml). Conversion of a r a c h i d o n i c acid to p r o s t a g l a n d i n E2 was confirmed by mass spectrometry. When s a l i n e washed t o a d f i s h red blood c e l l s were incubated w i t h a mixture of [ 1 - I 4 C ] - a r a c h i d o n i c acid and [ 5 , 6 , 8 , 9 , 1 1 , 1 2 , 1 4 , 1 5 , - 3 H ] _ a r a c h i d o n i c acid, comparison of the isotope r a t i o s of the r a d i o a c t i v e products i n d i c a t e d t h a t p r o s t a g l a n d i n F2a was produced by r e d u c t i o n of p r o s t a g l a n d i n E2. The c a p a c i t y of t o a d f i s h red blood c e l l s to reduce p r o s t a g l a n d i n E2 to p r o s t a g l a n d i n F2a was confirmed by i n c u b a t i o n of the c e l l s with [ I - 1 4 C ] p r o s t a g l a n d i n E2. The c a p a c i t y t o convert arachidonic acid to p r o s t a g l a n d i n s is widely distributed in mammalian tissues.

An exception is the mammalian erythrocyte

which appears to be devoid of cyclooxygenase a c t i v i t y ( I ) .

Recent evidence

indicates the presence of cyclooxygenase in tissues of teleosts and other vertebrate classes (2,3).

We now report that, in contrast to mammalian

erythrocytes, saline washed red c e i l s of the toad fish (Opsanus tau) have a high capacity to convert exogenous radiolabeled arachidonic acid to prostaglandin

E 2 and to prostaglandin F2a.

METHODS [ 1 - 1 4 C ] - a r a c h i d o n i c acid (55 mCi/mmol), [ 5 , 6 , 8 , 9 , 1 1 , 1 2 , 1 4 , 1 5 _ 3 H ] _ a r a c h i d o n i e acid (78 Ci/mmol), and [ 1 - 1 4 C ] - p r o s t a g l a n d i n E2 (40 mCi/mmol) were obtained from New England Nuclear (Boston, MA). Unlabeled arachidonie acid was purchased from Nuchek Prep (Elysian, MN). Authentic prostaglandin standards were the generous g i f t of Dr. John Pike of the Upjohn Co. (Kalamazoo, MI). Adult toadfish were purchased from the Marine Biological Laboratory (Woods Hole, MA). methanesulfonate (Ayerst) and blood was collected from the caudal artery into a heparanized syringe. Plasma was removed after centrifugation, and the blood c e l l s were washed three times with O.01M Tris buffer, pH 7.#, containing 0.9% NaCl and resuspended in the same buffer. In some experiments, a more complete separation of erythrocytes from cocentrifuging leukocytes was achieved by layering the blood over an equal volume of Ficoll-Paque (Pharmacia). When 0006-291X/83/01 0250-06501.50/0 Copyright © 1983 by Academic Press, Inc. A II rights o f reproduction in any form reserved.

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about h a l f of the blood c e l l s had s e t t l e d , the supernatant c e l l s and b u f f e r were removed, the bottom l a y e r washed three times w i t h T r i s - s a l i n e b u f f e r , and the c e l l s were resuspended i n the same b u f f e r . Aliquots of saline washed toadfish red c e l l s were incubated in Tris-saline buffer with [14 C]-arachldomc . . acld, . .a mzxture of [14C]- and [3H]-arachidonic acid, or [ 14C]-prostaglandzn . E2 at 37°C with shaking. At the end of the incubation, c e l l s were centrifuged, and the supernatant medium was drawn off, acidified with formic acid (final concentration I%), and extracted three times with an equal volume of ethyl acetate. After evaporation of the combined organic layers under a stream of nitrogen, the residue was redissolved in 50 ul methanol and applied to s i l i c a gel C/by plates (0.25 mm; Brinkmann), which were developed with the solvent system: ethyl acetate: iso-octane: acetic acid: w a t e r (110/55/20/~00; upper phase). Radioactivity was localized by radiochromatogram scanning (Packard 7220). In double isotope experiments, radioactive zones were scraped from the plate, suspended in 10 ml of Scintiverse (Fisher), and radioactivity was determined by s c i n t i l l a t i o n counting using a double isotope program (Beckmann LS7500). For determination of mass spectra, toadfish red c e i l s were incubated with unlabeled arachidonic acid (50 ug/ml). After removal of the c e l l s by centrifugation, the supernatent medium was desalted with a column of octadecylsilane (6 ml; Baker) as described by Powell (#). Prostaglandins were eluted with methyl formate. After evaporation of the solvent under a stream of nitrogen, the residue was treated successively with ethereal diazomethane, (15 min room temperature) methylhydroxylamine hydrochloride in pyridine (18 hr at 55°C), and N,O-bis (trimethylsilyl) trifluoroacetamide (3 hr at 55° C) and analyzed by gas chromatography-chemical ionization mass spectrometry using a column of 3% OV-I (235°C) f i t t e d to the i n l e t of a Finnegan 3200 Mass Spectrometer.

RESULTS AND DISCUSSION When [1-14C]-arachidonic

acid was incubated with a Tris-saline suspension

of human red blood c e i l s , no detectable conversion to radioactive products

occurs (Fig I A ) , c o n s i s t a n t w i t h the reported i n a b i l i t y erythrocytes to generate prostaglandins (I).

of mammalian

In contrast, when [I-14C]

a r a c h i d o n i c a c i d i s i n c u b a t e d with s a l i n e washed t o a d f i s h radioactive

fatty

red c e l l s ,

a c i d i s c l e a r e d from t h e medium by t h e c e l l

r a d i o a c t i v e products appear that p r o s t a g l a n d i n F2a (Fig I B ) . r a d i o a c t i v e products,

the

s u s p e n s i o n and

cochromatograph w i t h prostaglandin

E 2 and

Indomethacin (10 ug/ml) prevents formation of

but uptake of [ 1 4 C ] - a r a c h i d o n i c acid by the c e i l s

(data

not shown). To determine whether prostaglandin F2a was derived by reduction of prostagland{n E 2 or by direct reduction of the endoperoxide precursor,

p r o s t a g l a n d i n H2, a l i q u o t s of t o a d f i s h red c e i l s were incubated w i t h a m i x t u r e of

[1-14C]-arachidonic

(5).

acid and [ 5 , 6 , 8 , 9 , 1 1 , 1 2 , 1 # , 1 5 - 3 H ] - a r a c h i d o n i c

acid

Since 3H is l o s t i n s y n t h e s i s of p r o s t a g l a n d i n E2, t h i s product w i l l 251

be

Vol. 110, No. 1, 1983

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

A

o Origin

Arachidonlc Acid

B

/ 0 Origin

0

PGF2a

0

PGE2

Arachld0nlc Acid

Figure 1. Radiochromatogram scans of products recovered from the medium a f t e r i n c u b a t i o n of [ 1 - 1 4 £ ] - a r a c h i d o n i c acid ( l u C i ) with T r i s - s a l i n e washed human (A) or t o a d f i s h (B) red blood c e l l s f o r 1 hr at 37°C. A l i q u o t s of red blood c e l l s represented the red c e l l c o n t e n t of 5 ml (A) or 1 ml (B) or o r i g i n a l blood volume.

enriehed

in

14C r e l a t i v e

to

the starting

material

there is an isotope effect in removal of 3H).

(by 1.1#,

or greater

if

If prostaglandin F2~ is

produced by reduction of prostaglandin E2, it will also be enriched in 14C. Since no carbon-tritium bonds are broken in the synthesis of prostaglandin H 2 or reduction of prostaglandin H 2 to prostaglandin F2a , the latter process will yield prostaglandin F2a with the same isotope ratio as the substrate mixture, or enriched in tritium if there is an isotope effect in the synthesis of prostaglandin E2, favoring withdrawal of 14C from the endoperoxide pool (reference 5).

Since both prostaglandin E 2 and

252

prostaglandin F2a produced

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I I#C/3H R a t i o , Relative to Starting Material, of Products Incubation of Arachidonic A c i d w i t h T o a d f i s h Red B l o o d C e i l s Prostaglandln

Found

E2

Prostaglandin

1.19-+0.04

of (N=#)

F2e

1.12+0.01

> 1.14

Expected

by the toadflsh red cells were enriched in 14C (Table I), it appears that the latter product is primarily a metabolite of prostaglandin E 2 in these cells. Consistant with this finding, intact toad fish red cells are able to progressively reduce a pool of exogenous [14C]-prostaglandin E 2 to prostaglandin F2a (Fig 2).

Human erythroeytes contain an NADPH-linked

reductase that converts prostaglandin E 2 to prostaglandin F2~ (6), but the intact cells are unable to effect reduction of exogenous

prostaglandin E 2

(7), perhaps because of the inability of the latter to gain access to the cytoplasmic enzyme (8). Evidently the same permeability barrier does not prevent metabolism of exogenous prostaglandin E 2 by the intact toadfish red cell.

~

30 Seconds ...... ......

30 Minutes 120

Minutes

:/ ':. :~ ', : •, i .

."\ (..:

.

: :

".

•

Origin

i:

:: :: :~

~

:,

..,-

.I

i: ,:

..

.

- p

••Il •i

~

:,

'~.. ,-..."

".,.;,

.o

PGF2a

,....#.o..

.-.'".. ...

.

PGE2

Figure 2. Radiochromatogram scans of products recovered from the medium after incubation of [ 1 - 1 4 C ] - p r o s t a g l a n d i n E 2 ( 0 . 1 !JCi) w i t h a l l q u o t s of toadfish red b l o o d c e l l s f o r 30 see ( - ) , 30 min ( - - - ) , or 120 min ( ' ' - ) at 37°C. A l i q u o t s of red blood c e l l s c o r r e s p o n d e d to the red c e l l c o n t e n t of 1 ml of o r i g i n a l b l o o d volume.

253

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100-

0 225

1 O0

50

1

()0

150

277 295 366

]i ,l,l LT, I[

I.. +ll_ 200

..o

......

300

250

3,50

.

4()0

450

JL

I

,.

,..

500

L 550

.

+.

600

m/e XlO

100

525

277

(M+.+) 450

-

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+,, •

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200

250

300

350

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•

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m/e Figure 3. Chemical ionization (CH4) - mass spectrum (middle panel) of the major product recovered from the medium a f t e r incubation of arachidonic acid (167 uH) with toadfish red blood c e l l s corresponding to the red c e l l content of 10 ml of o r i g i n a l biood volume. The mass spectrum of authentic prostaglandin E2 (lower panel) i s shown for comparison. The toadfish red c e i l metabolite eluted from the 3% OV-I cloumn in 77 seconds (upper panel). Retention time of prostagiandin E2 standard was 77 seconds.

To confirm that the toadfish erythrocytes convert araehidonie acid to prostanoid products,

red c e l l suspensions were incubated w i t h unlabelled

arachidonic acid at a higher concentration (50 pg/ml) and the products analyzed by gas chromatography-chemical ionization mass spectrometry.

Only

one major product was detected, whose elution time from the OV-I inlet column and chemical ionization mass spectrum, were characteristic of prostaglandin E2 (Fig 3).

No prostaglandin

F2~ was detected in these experiments; it is

possible that the capacity of the red c e l l to generate this product is limited, and saturated at the high substrate concentration

254

employed.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The s i g n i f i c a n c e of t h i s metabolic c a p a c i t y of c i r c u l a t i n g erythrocytes, i n the f i s h ,

e i t h e r f o r the red c e i l remain to be determined.

itself It

or f o r c a r d i o v a s c u l a r f u n c t i o n s

is of i n t e r e s t t h a t human

e r y t h r o c y t e s undergo a measurable change in osmotic f r a g i l i t y to very low c o n c e n t r a t i o n s of p r o s t a g l a n d i n s ( 9 ) . to modulate the a c t i v i t y

toadfish

a f t e r exposure

P r o s t a g l a n d i n E2 i s known

of adenylate cyclase i n a v a r i e t y of t i s s u e s ,

i n c l u d i n g the r a t e r y t h r o c y t e (10,11).

I n any case, the g e n e r a l i z a t i o n t h a t

red blood c e l l s lack the c a p a c i t y to generate p r o s t a g l a n d i n s cannot be a p p l i e d to a l l

species.

ACKNOWLEDGEMENTS: We thank Dr. Dominic Desiderio f o r o b t a i n i n g the mass spectra. This work was supported i n part by a grant from the American Heart A s s o c i a t i o n , Tennessee A f f i l i a t e . REFERENCES I. 2. 3. #. 5. 6. 7. 8. 9. 10. 11.

H a r r i s , R.H., Ramweli, P.W., and Gilmer, P.3. (1979) Ann. Revs. P h y s i o l . #I, 653-658 Christ, E.3. and van Dorp, D.A. (1972) Bioehim. Biophys. Aeta 270, 537-5#5 Nomura, T. and Ogata, N. (1976) Biochim. Biophys. Acta #31, 127-131 Poweil, W.S. (1980) Prostaglandins 20, 9#7-957 Qureshi, Z. and Cagen, L.M. Bioehem. Biophys. Res. Commun. I04, 1255-1263 Kaplan, L . , Lee, S.C., and Levine, L. (1975) Arch. Biochem. Biophys. 167, 187 Smith, 3 . B . , S i l v e s , M.3., Ingerman, C.M., and Kocsis, 3.3. (1975) P r o s t a g l a n d i n s 9, 135-145 B i t o , L.Z. and Baroody, R.A. (1975) Am. 3. P h y s i o l . 229, 1580-158# Rasmussen, H. and Lake, W. (1977) in P r o s t a g l a n d i n s i n Hematology, M.3. S i l v e r , 3.B. Smith, and 3.3. Kocsis, eds., Spectrum, pp. 187-202 Sheppard, H. and Burghardt, C.R. (1970) Mol. Pharmacol. 6, #25-#29 Rasmussen, H., Lake, W., and A l l e n , 3.E. (1975) Biochem. Biophys. Acta #11, 63-73

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