Transformation of arachidonic acid in the rat anterior pituitary

Vol. 104, No. 3, 1982 February 11, 1982

BIOCHEMICAL

TRANSFORMATION

OF ARACHIDONIC

Sylvie

d'Endocrinologie 1'Universits

Received

December

ACID

Pierre

Pilote,

Laboratoire

AND BIOPHYSICAL

16,

Laval,

RESEARCH COMMUNICATIONS Pages 867-873

THE

RAT

Vallerand

and

Pierre

MolPculaire, Quebec

GlV

IN

ANTERIOR

PITUITARY

Borgeatl,

Le Centre Hospitalier 4G2, Canada

de

1981

Summary: Rat anterior pituitaries were incubated with [l-14C]arachidonic acid. The metabolites were purified by reversed-phase high pressure liquid chromatography. Conclusive identification of the compounds was performed by gas chromatography-mass spectrometry. The major metabolite of arachidonic acid was the 12-hydroxy5,8,10,14-icosatetraenoic acid (0.1% of added radioactivity). Smaller amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid and of 15-hydroxy-5,8,11,13-icosatetraenoic acid (0.01% of added radioactivity) were also isolated. Trace amounts of prostaglandins E2, D2 and F2a were detected.

It

has

been

shown

marked

elevation

of

tissue

and

the

cause

arachidonic

acid,

substance

9).

These

findings

acid

might

be

for

first

step

acids

in

'Scholar

on

CAMP

here

of

growth

the

there

is

the

isolation

the

Medical

PGE2,

release

role on of

some

at

the

and

Research

of

CAMP in

pituitary

(l-7).

Moreover,

mimics

role

the

of

vitro

(8,

arachidonic the

anterior

no

direct

evi-

this

system.

As a

CpO-polyunsaturated

of

adenohypophyseal

structure

identification,

Council

of

from

time in

a

effects

of

hormone

present

induce

in

metabolites

icosanoids

release

PGE2

GH release

of

the

of

and

hormone

and

that

in

studies

mechanism

of

accumulation

a physiological

the

of

suggest

our

PGEl level

precursor

involved

in

that

intracellular

release

however,

dence

report

the

the

this

pituitary;

previously

of

fatty hormones, by

we

physi-

Canada.

Abbreviations used: PGs, prostaglandins; CAMP, 3',5'-adenosine cyclic monophosphate; HETE, hydroxy-icosatetraenoic acid; HHT, 12-hydroxy-5,8,10-heptadecatrienoic acid; HPLC, high pressure liquid chromatography; GC, gas chromatography; MS, mass spectrometry; UV, ultraviolet.

867

0006-291X/82/030867-07$01.00/0 Copyright 0 I982 by Academic Press, Inc. All rghts of reproduction in any form reserved.

Vol. 104, No. 3, 1982 cochemical in

the

rat

BIOCHEMICAL

methods,

of

the

anterior

pituitary.

AND BIOPHYSICAL

major

MATERIALS AND PROCEDURES [1-14C] arachidonic acid arachidonic acid and purified before use. PGs were obtained Michigan); 5S-HETE, 15S-HETE, biosynthesis from arachidonic and purified by HPLC (details separately).

metabolites

RESEARCH COMMUNICATIONS of

arachidonic

acid

was diluted to 30,000 CPM/ug with by silicic acid chromatography from the Upjohn Company (Kalamazoo, 12S-HETE and HHT were obtained by acid and appropriate enzyme sources, of these procedures will be reported

Preparation of pituitary tissue. Male Sprague-Dawley rats weighing 120-150 g were sacrificed by decapitation and the anterior pituitary was removed and placed within 30 set into ice-cold Dulbecco's phosphate buffered saline (without Ca++ and Mg++) containing 1 mM of sodium ethylene diamine tetraacetate. Intact pituitaries (lo/2 ml) were washed 3 times during 5 min at 2°C. Incubation. The pituitaries were then warmed to 37" and pre-incubated during 10 min; the medium was then changed for normal Dulbecco's phosphate buffered saline (10 pituitaries/ml). [l-14C]arachidonic acid was added (in solution in ethanol) to a final concentration of 15 pg/ml (the maximum final concentration of ethanol was 0.2%). After 10 min of incubation, 1.5 volume of methanol was added. Extraction and fractionation. The denaturated incubation mixture was homogenized and centrifuged (2000 g x 5 min); the pellet was homogenized again with 0.5 incubation volume of methanol. After centrifugation, the two supernatants were pooled, acidified (pH 3.5) with citric acid and extracted with diethyl ether as described previously (10). The ether extract was fractionated on a silitic acid column (0.5 cm diameter glass column packed with 1 g of Si1ica.r CC-4, Mallinckrodt) by successive elutions with 30 ml of diethyl etherlhexane, 15185, v/v, 30 ml of diethyl ether/hexane, 45155, v/v and 30 ml of diethyl ether/methanol, 92/8, v/v. The first fraction contained the unreacted arachidonic acid and other unpolar lipids, the second fraction contained the HETEs and HHT, and the third fraction contained PGs and other polar lipids. HPLC (Radial Pak C18, was performed using radially compressed columns 100 x 8 mm, 10 u particles from Waters Associates, Milford, Mass., The fractions "45/55** and '*92/8" (see above) were evaporatUSA). with diazomethane and analyzed (HPLC) using respeced, esterified The tively, methanol/water, 75125 and 65135 (v/v) as solvents. elutions were monitored by ultraviolet photometry and radioactivity detection (Berthold HPLC Radioactivity monitor model LB503). Radio-GC and GC-MS analysis. Radio-GC was performed using a Varian 3700 GC coupled to a Packard radioactivity detector, model 894. The column'used (glass column, 2 m long,-4 mm inside diameter) was packed with SE-30 1% on Chromosorb-W-HP. Argon was used as carrier gas. GC-MS analysis of the methyl ester trimethylsilyl ether derivatives of the compounds were performed as described before (11). RESULTS Fifty with

pituitaries

arachidonic

(about acid

as

400

described

mg of above.

868

tissue)

were

Measurements

incubated of

the

Vol. 104, No. 3, 1982

BIOCHEMICAL

6

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12-HETE-Me

48

40

24

32

ELUTION

TIME

la

a

0

(min)

Fig. 1. Reversed-phase HPLC chromatograms of the -45/55* fractions (treated with diazomethane) obtained from the silicic acid chromatography of ether extracts. Intact rat pituitaries were incubated with arachidonic acid (A); rat pituitaries were boiled during 5

min before incubation with arachidonic acid (B) Arrows indicate the positions of various ment). under identical conditions in a separate analysis. formed as described under Materials and Methods.

radioactivity

content

chromatography of

In

control of

but vity. that sample

the

HPLC

the HPLC

three

analysis the

corresponded

and the

arachidonic than

traces

one

of

the

"45/55"

showed of

to

HHT,

869

and radioac-

were

boiled

fractions

5 min

-45155"

and

radioactivity.

detectable

and

99.5%

recovered

after

UV absorption

absorption

15-HETE

the

acid

-45555"

fraction

hydroxy (UV

of

recovered

of

some

various

compounds

and

0.05%

peaks

fractions 0.2%

silicic

afforded

pituitaries

acid

of

from

"15/85" the

0.3

Several major

of

fraction whereas

less

diazomethane. only

fractions

experiments,

'* 9 2 I 8 '* contained

with

that

respectively

addition

1A shows

three

radioactivity,

" 9 2 / 8 " contained

before

the

indicated

recovered

tivity.

of

(control experistandards eluted HPLC was per-

acids

treatment

were amount

of

standards peaks)

12-HETE.

Fig.

detected HPLC

present radioactisuggested in analysis

the of

Vol. 104, No. 3, 1982

BIOCHEMICAL

b

AND BIOPHYSICAL

h

b

;*

ELUTION

Fig.

2.

RESEARCH COMMUNICATIONS

TIME

i6

i.

(min)

Radio-GC

analysis of the major metabolite (methyl ester, ether) of arachidonic acid in intact rat anterior The chain length equivalence (with saturated fatty esters) of the material eluting at 10 min was 21.3 RAD, radioactivity FID, flame ionization detector;

trimethylsilyl pituitaries. acids methyl carbon units.

detector.

the

"45155"

tissue)

did

tivity

peaks

material

fraction

obtained

not

the

(Fig.

of

21.3

acid

identity

of as

-4/555**

material was

also

with

the

two

(12,

units

UV absorption analysis

of

with

and

the

was

(not

compound

UV absorption analyzed

by

hydroxy

Although

peaks

performed:

length

shown)

and

the

was

confirmed

the

mass HPLC

spectra analysis 870

icosa-

confirmed

present

in

corresponding identity

of

of

those the

fraction

The

HHT and

the

compounds

of

of the

the

fraction

to

by comparison with

the

equivalen-

acid.

GC-MS;

acids

radioac-

a monohydroxy

analysis

radiolabeled

(boiled

radiolabeled

a chain

supporting

GC-MS

the

standard

a peak

(C-21.3)

experiment

12-hydroxy-5,8,10,14-icosatetraenoic

equivalences 14).

control

12-HETE

showed

major

the

in

the

(12-14).

the

HETE

length

2)

carbon

tetraenoic

Radio-GC

with

(Fig.

the

corresponding

1B).

comigrating

chromatogram ce

show

from

15-

chain

standards W45/55M

did

Vol. 104, No. 3, 1982 not

show

and

HHT

two

compounds

method The

any

detectable

(because

for

the

of

found

to of

was

compounds step.

same

lyzed

small

leukotriene

preliminary the

the

"92/S"

various

increased

D2 and

sample

The

using

radiolabeled (liquid the

B,+ (data

not

shown).

the

were

on was

Fractions

multiple

ion

tissue

The was

a more

sensitive

(l-10

thrombo-

elution

and

time in

using were

technique

not

of a

eluted

interest

ng/50

(control

PGs,

determined

of

PGs were

boiled

the

of

detection

amounts

15-HETE

scintillation).

injected

system.

the

present),

presence

HPLC

then

detected.

from

using

for

sample

with

material

analyzed

Small

F2u

obtained

be

(standards)

sensitivity.

PGE2,

amounts

radioactivity

chromatographic

by GC-MS

RESEARCH COMMUNICATIONS

associated

measurement

Bp and

AND BIOPHYSICAL

radioactivity

of were

fraction

xane

BIOCHEMICAL

ana-

for

pituitaries)

of

detectable

in

the

experiment).

DISCUSSION In

this

paper

arachidonic

acid

bolites ty

were

of

we in

rat

isolated chromatographic

chain

length

equivalences

mass

spectra,

mistry

of

the

amount

of

material

matic

formation

ments

where

rest.

The

14)

3).

(Fig.

could of

be

from

tempts

was

the

to

increase

could

added

1% in the

the

be

formed

formation

transformation,

from

871

because

in

inte-

PGs by

PGs)

(12,

(15)

(14).

The

rat

pituitary

performed). the

pathway

and

of acid

of

a lipoxygenase

a lipoxygenase

enzyexperi-

compounds

(HHT

the

the

control

by

i.e.

and

however,

the

experiments

similari-

stereoche-

determined

pathway

arachidonic all

The

assessed

meta-

HPLC,

standards.

produce derive

the

chromatography)

was

not

of

reversed-phase

not

of

Several

basis

unsufficient;

cyclooxygenase

in of

(below

did

identified

a by-product

low

was

metabolism

by gas

was

compounds

tissue

15-HETE

transformation

sue

authentic

identified

these

compounds and

of

the

on

(determined

the

tissue.

on

behaviors

those

on

pituitary

identified

available of

studies

anterior

compounds

boiled

(12-HETE)

some

and

their

with

report

percentage

Every modification

or

tisatof

Vol. 104, No. 3, 1982

8lOCHEMlCAL

AND 8lOPHYSiCAL

RESEARCH COMMUNICATIONS

LlpoxyG~~~xyG [12-HPETEI

IPGG,l-

[15-HPETE]

PGD, PGF$

1

\

I

8c-q

$.g+

GO”

12-HETE

OH

15-HETE

HHT

Fig. 3. Metabolites of arachidonic acid identified in rat anterior pituitaries (intact tissue). Compounds between brackets have not been isolated (see Discussion). The stereochemistry of the HETE’s has not been determined in these studies but was assumed to be identical to that of the same compounds previously obtained from other sources (12. 14).

incubation

conditions

bation,

addition

homogenates, tissue

of were

in

tissue could

bolites

be

A23187)'

studies,

acid.

in

it

or

small

amount

the

seems

in

that

whether

part)

anterior

the

of

pituitary

this

of

tissue

pituitary

PGs

to

found

not

in

these

vascular

meta-

tissue

rather

possibility

incu-

capacity

or

from

of

use

a limited

as

least

the

that

has

question

(at

present cells;

The

the

derived

duration

suggesting

these

raises

fibroblasts

hypophyseal

than

from

could

not

be

insight

to

the

out. Nevertheless,

the

biochemistry

of

results

point

pathway

in

way).

ionophore

arachidonic

pituitary

ruled

the

concentration,

unsuccessful,

obtained

as

metabolize

or

(substrate

Thus,

lipoxygenase

arachidonic the

out

this

present

will

(as be

products

2The compound A23187 release of arachidonic tems, and activates

acid

relative

tissue it

studies

on is

of the

bring in

the

a new anterior

importance

of

compared

to

the

interest

to

study

release

a cation acid and the leukocyte

of

the

the

C-12

lipoxygenase

cyclooxygenase

path-

the

hormones

ionophore the synthesis lipoxygenase 872

pituitary:

which

effect from

of the

various pituita-

stimulates the of PGs in many sys(16, 17).

Vol. 104, No. 3, 1982 ry, HETE

and

also

to

in

this

tissue

BIOCHEMICAL measure

the

under

AND BIOPHYSICAL changes

of

RESEARCH COMMUNICATIONS

concentration

experimental

various

of

the

12-

conditions.

REFERENCES 1. 2. 3. 4. 5. 6.

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Labrie, N-3 and Physiology, Bergeron,

F., Borgeat, P., Ferland, L., Drouin, J., Barden, Beaulieu, M. (1980) Prostaglandins and Reproductive INSERM Publications 91, 19-44. L. and Barden, N. (1975) Union Med. Canada 104,

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Borgeat, P., Picard, S., Vallerand, P. and Sirois, P. (1981) Prostaglandins and Medicine 6, 557-570. B. (1974) Proc. Nat1 Acad. Sci. Hamberg, M. and Samuelsson, U.S.A. 71, 3400-3404. Borgeat, P., Hamberg, M. and Samuelsson, B. (1976) J. Biol. Chem. 251, 7816-7820. Wlodawer, P. and Samuelsson, B. (1973) J. Biol. Chem. 248, 5673-5678. Narumya, S., Salmon, J.A., Cottee, F.H., Weatherley, B.C. and Flower, R.J. J. Biol. Chem. 256, 9583-9592. Pickett, W.C., Jesse, R.L. and Cohen, P. (1977) Biochim. Biophys. Acta 486, 209-213. Borgeat, P. and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA 76, 2148-2152.

873