Arachidonic acid metabolism in guinea pig megakaryocytes

Vol. 107, No. 2, 1982 July 30, 1982

AND BIOPHYSICAL

BIOCHEMICAL

Pages

ARACHIDONIC

ACID

METABOLISM

IN

GUINEA

I Jonathan

L.

Miller

, Marie

J.

PIG and

Stuart?,

+

Received

June

t

Departments of Pathology and Upstate Medical Center, Syracuse,

SUNY

RESEARCH COMMUNICATIONS

21,

752-759

MEGAKAPYOCYTES Ponald

W. Valenga+

Pediatrics New York

13210

1982

SUHHARYlqGuinea pig megakaryocytes were isolated to 75-90% purity, incubated with C-arachidonic acid and radioactive metabolites analyzed by thin layer chromatography. Thromboxane B l2-hydroxyheptadecatrienoic acid and 12-hydroxyeicosatetraenoic a?i’d (I2-HETE) were the major products (HHT) identified. Pre-incubation with indomethacin inhibited production of thromboxane B2 and HHT. Comparison of metabolites produced by megakaryocytes and platelets from the same animals indicated that one megakaryocyte produced 2500-4000 times as much thromboxane B,, HHT. or 12-HETE as did a sinale platelet. These resul ts suggest that z i sol ated megakaryocytes possess the full complement of cyclooxygenase and lipoxygenase activities found in circulating platelets. Arachidonic and

acid

secretory

of

inhibitors

of

thromboxane

of

treatment

drug

regimens it

of

the

suspensions

of

the

have

and

becomes

in

and

direct

and

processes

in

assessing

critical

from and

in

the

beginning

This

work pig activities

the

aggregation

Indeed, work

on

the

medicine to --in

vivo as

(2).

to cells

utilizes

highly

marrow of

bone

for

the

In

both

administration

precursor

bone

use

inhibitors

implications

information the

in (I).

significant

guinea

lipoxygenase

role

platelets

clinical

have

metabolism

megakaryocytes

important

response

to

megakaryocytes.

cyclooxygenase

an blood

cyclooxygenase

arachidonate

platelets,

both

platelet

plays

circulating

thromboembolic

agents,

pathways

of

synthetase of

designing such

metabolism

responses

the

to marrow

of operative of

blood

purified characterize megakary-

ocytes. MATERIALS AND HETHDDS Preparation of Megakaryocytes and Platelets. Albino guinea pigs (Charles River) of either sex (300-450 g) were anesthetized with nembutal (50 mg/kg). Blood taken by cardiac puncture was anticoagulated with The animals were then 0.38% sodium citrate and washed as described below. sacrificed and the humeri, femurs, and tibia removed. Marrow was removed from these bones and processed by minor modifications of the method of Levine and Fedorko (3). In brief, marrow was taken into calciumand magnesium-free 1 mM Hanks ’ balanced salt colution (CMFH) containing 0.38% citrate, adenosine, and 2 mM theophyl I ine, washed, and layered onto a discontinuous gradient of bovine serum albumin (Fraction V BSA, Armour ) for density gradient centrifugation (adjusted by refractive index, with ascending indices (at 20°C) of 1.3694, I .3662, 1.3630, and 1.3598). After 30 min. at 10,000 x cells remaining above the pellet were pooled, washed in CMFH, and g, 4Oc, all layered on a BSA velocity sedimentation gradient of I.3430 bottom layer, 2:1 (v/v) of 1.3430: CMFH middle layer, and I:2 (v/v) of 1.3430: CMFH top layer. 0006-291X/82/140752-08$01.00/0 Copyright 0 1982 b-v Academic Press, Inc. AN righis of reproduction in any,form reserved,

752

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

After 30 minutes at 20°C and unit gravity, cells above the second highest interface were discarded and the remaining cells washed in CMFH and applied to a second velocity sedfmentation gradient. After discarding the top layers once again, the remaining cells were washed in CMFH, counted, and studied. Isolated marrow cells from each animal were counted in six hemocytometer chambers by phase contrast microscopy. Oifferential counts of megakaryocytes versus non-megakaryocytes were performed upon Wrights-Giemsa stained cytocentrifuge (Shandon Corp.) preparations, in order to maximize discrimination between the smaller megakaryocytes and cells of other I ines. Total megakaryocytes as we1 I as megakaryocyte purity were calculated by mul tiplying the differential counts by the total cell counts obtained by phase contrast. Citrated guinea pig blood and human blood (drawn by venipuncture from volunteers giving informed consent) was centrifuged for 20 min. at 100 x g to produce platelet-rich plasma (PPP). The ccl Is were pelleted at 4’C in the presence of 1% EDTA and resuspended in CMFH. The procedure was repeated with successively decreasing concentrations of EDTA, until the platelets were firally resuspended in CMFH alone. The final platelet suspension was counted either by phase contras,t or electronically (Model S Plus, Coulter). Metabolism of l- ‘7C-Arachidonic Acid and Analyses of Metabolites. l-‘4C-arachidonic acid (50-58 Ci/mol) was purchased from New England Nuclear or Amersham an@ stored as an ethanol solution at51-5 mM. Cells were suspended at 2-5 x 10 platelets/ml or 0.5-2.5 x 10 megakaryocytes/ml in CMFH . . contatnrng 25 mM Trrs-HCI, p H 7.4. Suspensions were incubated at 37OC for 5 mirlutes (,in the presence or absence of 30 pM indomethacin), made 1 .O mM in CaC 1 anld arachidonic acid was then added to a final concentration of I to 30 -J: Cells were incubated at 37’C with gentle shaking for the indicated times. Metabolism was terminated by the addition of three volumes of a I :2 (v/v) mixture of chloroform:methanol. Metabolites were extracted essentially as described previously (4). Thromboxelne B2 (TXB ), prostaglandin E , prostaglandin 0 and prostaglandin Fla (PL Eliochemicalf, 12-HETE (New Eng f and Nuclear), 5-Hg;E (a gift from Dr. P. Borgeat) and 15-HETE (prepared with soybean I ipoxygenase) were used as internal standards. Three volumes of chloroform were added, and the extract was, acidified with 0.2 volumes of 0.5 N HCI. Phases were thoroughly mixed, then separated by centrifugation. The lower phase was removed, dried over anhydrous, magnesium sulfate and evaporated in conical vials under a stream of n i trogen. The extracts were analyzed by thin layer chromatography on Silica Gel G in chloroform:methanol:glacial acetic acid:water (90:8:1:0.8 v/v/v/v) (System A) which resolves all metabolites of human platelets (5); or ether:petroleum ether:glacial acetic acid (100:50:1 v/v/v) (6) (System B) which resolves positional isomers of hydroxy fatty acids (7). Extracts were also analyzed by conversion of free fatty acids to methyl esters with diazomethane (8) and separation in System C (upper phase of water:ethyl acetate:iso-octane Chromotograms were analyzed by radioautography on (100:50:100 v/v/v)) (9). Kodak XAP film developed in an X-Omat processor. Incorporation of radioactivity into metabolites was measured by scraping regions of the chromatogram indicated by radioautography and liquid scintillation counting of the scraped adsorbent in 83% Aquasol (New England Nuclear), 12% water, 5% methanol. RESULTS

Meqakaryocyte

tation

steps,

animal,

at

ccl

volume of

the

Due

to

the

greater marrow

be higher

l-m3

for

Purity.

still. megakaryocytes

By

yields

purities

contaminating

would

15,000

and achieved

megakaryocyte

I number.

wi th

Yields

we routinely

of

ranging average cells,

utilizing I .4-5.0

from

75 to

size

of

megakaryocyte

Utilizing

the and

753

sedimen-

90% of

ccl

volume pm3

nucleated

megakaryocytes purity

relative 420

two velocity x 10 5 megakaryocytes

for

as in

terms

estimates non-megakaryocytes,

per Is

by

compared of

ccl

(10,ll)

I

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

A

RESEARCH COMMUNICATIONS

B

4

-ID Fiqure 1 - Products of arachidonic acid metabolism megakaryocytes. Autoradiographs of thin layer (plate A) or system B (plate 6). On each plate were guinea pig megakaryocyte products, guinea human platelet products. Presumptive identity acid; (2) 12-HETE; (3) HHT; (4) TXE2.

megakaryocyte

cell

mass

in

our

studies

by guinea chromatograms the samples pig platelet of bands:

would

comprise

pig

platelets and in system A from left to right products, and (I) arachidonic run

over

99%

of

total

cell

mass. Identification ocytes with

platelets

included

a

(Fig.

Arachidonic

Acid

produced

14 C-arachidonic

l-

bands

of

of

and

a

acid

band

which

which

migrated

IA).

The

slower

the

faster

of

the

of

two

two

migrated

with (Fig.

‘s :B)

yet

unidentified, HHT

platelets.

(0.37

and

and

and

platelets.

positional

an

were

0.49)

distinct products also These Minor

metabol

Rf

human

mobili

ties

i tes

did with

in not mobi

correspond Ii

ties

to In

from

Tar

of B,

12which

Addi

platelets tional,

from

as 12-HETE

megakaryocytes

major to

that

migrated

human

slightly pig to

simi

system

products

prostaglandins

A

The

that

products

(0.34).

guinea

to

platelets.

major

differing

two

system

identical

human

l2-HETE

both

in

identical

5-HETE

and

standard

0.71,

of

two

and or

products

chromatogram

platelets.

HHT

incubated

radioactive

by

these

(0.52)

observed

Rf

0.78,

of

to

with

an

megakary-

when

internal the

produced

HETES,

Is-HETE

routinely

components

of

identical

from

of

with

from

isomers

TX62

(HHT)

(I2-HETE)

even R

with

the

pig

products

major

region moved

acid

of

The

with

these

hydroxyeicosatetraenoic resolves

I).

hydroxyacid

12-hydroxyheptadecatrienoic

Guinea

spectrum

(Fig.

co-migrated in

Metabolites.

similar

and of

human O2

and

Vol. 107, No. 2, 1982 Table

BIOCHEMICAL

1.

Effect

Acid

of

AND BIOPHYSICAL

lndomethacin

Metabolites

by

On

Guinea

Pig

the

Production

34

Arachidonic

and

Platelets’

HHT

42

+ 9.8

8.8

Platelets

of

Megakaryocytes

TXB2

Megakaryocytes

RESEARCH COMMUNICATIONS

+ 2.6

18.2

12-HETE

+ 4.6

98

+

17.3

-+ 4.4

110

+

36.3

* Suspensions 37°C

for

the

addition

are

analyzed

)

in

A

system

compounds met hy I

esters

mepakaryocytes

with

as

‘S were

of

these

were

also

(5 the

$IM final

in

major

text,

0.5) internal

tical

solvent

from those

HHT

was

of

an were

system

-+ S.E.

for

co-migrate

standards.

to

to

After

(Mean

not

at

prior

products

using 100.

did

products

iden

incubated

concentration).

x

as

were

I.IM indomethacin

precursor,

the

and

included

30

radioactive

described

0.4

platelets

of

(CPM,ndo/CPMContro,)

between

they

or

absence

with

The

guinea

pig

human

platelet

A. three

mobil

these i ties

of

platelets

and

products

in

C. The

guinea

identification

pig

of

platelets

pM

indomethacin.

in

human

or

p;g

80%

inhibited

inhiloi

tion

of

respectively), Kinetic with

5 uM up

high their

tc’ rate

Analyses l-

of

for

(Fig.

fifteen

one

were Five

similar

to

minute

production

of

megakaryocytes half-maximal

all from

the

megakaryocytes

exogenously production

some

of

were i tes.

both the

resul

ts In

and

TXB2

seen

these in

which

with

30

and

HHT

lipoxygenase

were

HHT

lncuba

a

linear

for

the

experiments platelets

(over

58%

and

pig

tion

of

inhibition

of

were

time

more

approximately

acid

while

of

metabolism. rate

thromboxane

appeared 5

uM

all

though

initial of

a in

of even

course

HHT

variable

remained,

approximate

production

and at

production

acid

arachidonic at

TXB2

continued

arachidonate

their to

The

of

terminated

arachidonic in

megakaryocytes

l2-HETE

platelets

addition

selected

of

accumulation

animals

provided occuring

of

(66%

production

Guinea

non-metabolized

metabol

to The

of

incubations

TXE2

5 minutes

of

1).

in

12-HETE.

led 2).

from minute

quantities

both

Production.

minutes. Platelets

within

significant

of

acid

minutes

ion

megakaryocytes

tion

Metabolite

C-arachidonic

five

metabolism.

prcducts others

14

(Table

in

for

product

similar

of

and inhibi

experiments

production

i tatively

production

both)

without

the

inhibit

megakaryocytes

the

by

preincubated

ts

not

Qdal and

supported

were inhibi

does

(12,13).

platelets

indomethacin

and

compound but

I2-HETE

guinea

TX82

megakaryocytes

This platelets,

prcduct

with

or

acid

as

(Pf

when

megakaryocytes

incubation

expressed

experiments.

pig presence

I-‘4C-arachidonic

and

Results

for

the

5 minutes

extracted

system

guinea in

of

additional

E2

of

5 minutes

of by

saturable, (Fig.

3).

By

Vol.

107,

No.

BIOCHEMICAL

2, 1982

AND BIOPHYSICAL

0

5

IO

TIME Figure 2 megakaryocytes

acid

for

-

the

and (A) experiments.)

contrast,

indicated

of

the

between by

acid

3 and

was

of

up to

of

acid

did

animal

arachidonate,

Guinea

5 uM

incorporated (Data represent

saturation

produced,

metabolism.

with

incubated

Similar

suggested

15

(Minutes)

considerable

products pigs

10 pM

l2-HETE

30 PM. Vhile

guinea

arachidonic were Radioactivity determined.

10’)

times. (0)

platelets.

amount

individual

of x

production

concentrations pig

course - 1.0

I2-HETE

the

guinea in

Time (0.5

RESEARCH COMMUNICATIONS

data

that

thromboxane

while

I2-HETE

not

into

appear kinetics to

animal

obtained

pig

i-14C-arachidonic TX82 means

(o), of

HHT two

to

saturate

were

observed

platelets

production

was

production

for existed

variation

with

at

was

not

from saturable sat urated

30 uM arachidonate. In

an

effort

by

guinea

to pig

compare

platelets

the

relative

and

their

amount progenitor

HETE (0)

determined.

of

metabolism

arach

idonic of

30

ARACHIDONIC

dependence the indicated the incorporation (Mean + S.E.

metabolism cells,

IO

I35

Figure 3 - Concentration were incubated with for five minutes and

of

ACID

(‘,M)

of metabolite productlion. Megakaryocytes C-arachidonic acid concentrations of lof radioactivity into TXB2 (0) and 12for four experiments.)

756

5 UM

Vol. 107, No. 2, 1982 Table

2.

BIOCHEMICAL Relative

AND BIOPHYSICAL

Production

Metabolites

by

of

Guinea

Pig

Cyc’ooxygenase

and

Hegakaryocytes

TXB2

RESEARCH COMMUNICATIONS

and

HHT

Lipoxygenase

Platelets.’

‘2-HETE

-Megakaryocytes

3.2

+ 0.7

Platelets

8.3

+- 2.0

10.0 39

-+ 2.6

15.8

-+ 7.4

48

2.0

(pmoles/lO

+ 9.2

(pmoles/lO

+

5 cells) 8

cells)

-1

Megakaryocytes

and

5 !.IM I-‘4C-arachidonic from

the

acid

specific

endogenous

megakaryocytes

isolated

suggest

that

TXB2 3 3900

each as

We have

the

and

the

megakaryocytes study

of

chromatographic

with

authentic

products inhibitor acid

cyclooxygenase) tes

marrow

The

preseplt

as

a

of

studies,

the

capaci

TXB2

radiolabeled

is

as

than

that

shown of

in both

acid

The

various and

on

additional

the

one

Table l2-HETE

2,

the and

in

of

several

is

incubated

relative

the

151

TXB2

various

selective arachidonic

from

these

and

cells

(14).

techniques,

as

the

use

moreover, products

isolated

well

megakaryocytes

Through

of

the

Worthington by

work,

identifiable with

well-defined (a

that

ion. present

basis

identification.

isolation

production

HHT.

the

the

confirmation

product

employed

to

the

megakaryocytes

of

different provide

and

pathways

on

purity),

production

thromboxane

acid

purify

megakaryocyte

quite

only

of

seen

co-chromatography

with

for

guinea

megakaryocyte

indomethacin

used to

final

species,

types.

synthesis

much

pathways

the

established

of

criterion

of

was

identity

of

synthetic

systems,

effects

as

2

platelet.

thromboxanes

cell

solvent

The

for

purity

products

each

major

yield

these

Table

times

as

the to

of

in

preparation

via

high

and

platelets

2500 I2-HETE

cyclooxygenase

the

employing

ty

of

unidentified

presented

purified

contaminating

the

arachidonic

fact,

to

(S-IO%

calculated

in

data

much

attribution

in

techniques

that

--

described

animal

The

as

elutriation

rats

with

contribution

include

approximately

I2-HETE.

(13)

an

recently

chromatographic

clear

a highly

platelets.

was

different

possess

that

of

centrifugal

have

times

standards,

of

pmoles

studied

animals.

produced

purified human

Utilizing

Nakeff

3000

identity

of

metaboli

femoral

and

mobility

may

was

same

than

incubated

any

values

minutes

unequivocal

the

and

provided,

HHT

arachidonic

rather

this

were

analyzed,

the

yield

pigs

experiments.)

platelets to

allows

In

5

HHT,

human

lipoxygenase

preparation

for

metabolizes

pig

and

eight

demonstrated

megakaryocytes guinea

for

guinea

acid

ignored.

from

much

same

products

megakaryocyte

times

DISCUSSION! in

S.E.

the

arachidooic

being

acid

I-‘4C-arachidonic

HHT:

the

acid +

from 5 minutes,

of

(Mean

ac ids.

pig

for

activity

arachidonic

hydroxy

platelets

it

of is

formed

when

megakaryocytes. TXB2

appears

the now

In to

be

less

Vol.

107,

No.

The

ability

pathways

in

to

both

platelet

study

speculation the

as

to

of to

suggests well

TX6

that as

the

with

other inhibitors

study,

cultured

in as

vitro

to

acid

studies

as

well

karyocytes

(21),

are

contain

the

necessary

full

for

the

the

in

per

to

suggest

that

complement

of

eventual

administered

I of

immediate - in This

with

platelets,

as

of and of

their

a subject

megakaryocytes

cyclooxygenase

inhibitors

cf

values

of

for

such

baseiine prove

effects

upon

useful megakaryocytes

of

(19,20). and

tional

lipoxygenase progeny,

ly,

to

the

the that

per

we

have

of

of mega-

metabo-

platelet.

Since

1000-5000

and

ratio for

production as

approximately

cyclooxygenase

metabolites as

similar

megakaryocytes

platelet

was

interest,

their

produce

ccl

an

susceptibility

megakaryocyte

the

considerable

progenitor

abi I ity

was

Addi

great

of

megakaryocytes.

should

--in vivo cyclooxygenase

2).

as

estimated

their

of

(1:4.7:5.7)

(Table

results

further

paper

and

platelets

times

common Of

these

estimation

found

share

by

present

megakaryocytes

2500-4000

these

of

pig

of

cyclooxygenase

the

the

establishment

involving

proportions

megakaryocytes

the

studies

(1:3.1:4.9) was

have

inhibition The

administered

for

a

type.

from

in

TXB2:HHT:12-HETE

this

recover

metabolism

in

on

We

animals

determination

aspirin.

relative similar

lites

the

for

these

of

generated

have

guinea

tot-s

for

indomethacin

of

studied, of

inhibitors

The appear

when

from

or

as

cyclooxygenase

has

may

production

use

aid

(l7,16).

cells

is

indomethacin

arachidonic

agents

megakaryocytes

inhibi The

(16)

suspensions

to

continuing

such

HHT

most

cyclooxygenase

such

metabolic

a diagnostic

production

effect

RESEARCH COMMUNICATIONS

useful.

as

megakaryocyte

and 2 purified

highly

of

prove and

and

the

the

inhibition

should

(IS)

platelet,

vitro

effects

therapeutically

survival

AND BIOPHYSICAL

the

megakaryocytes

inhibitors

of

BIOCHEMICAL

2, 1982

platelets

studied

lipoxygenase

already activities

progeny.

ACKNOWLEDGMENTS The Suchomsk

authors i ,

supported

for by

their

to

thank

Health

Dusse,

technical

Research

Manufacturers to

John

excellent

N.Y.S.

Pharmaceutical HD-14405

wish

Carolyn

Ganley,

assistance.

Council

Association

This

II-037

Grant

and

Foundation

to

research

and a grant JLM,

and

from USPHS

MJS. PEFEPENCES

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A.J.

2.

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J.B.

3.

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P.

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Am.

Fedorko,

T.K., Smith, 424, 303-314. J.Y.,

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P.k’.,

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BIOCHEMICAL

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Renkc’nnen,

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Walenga, Life Sci.

R.W., 27,

Schlenk,

H.

and

Gellerman,

M.

and

Samuelsson,

8.

0. (1966)

AND BIOPHYSICAL

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Showell,

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H.J.,

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RESEARCH COMMUNICATIONS 288-309.

125,

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and

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(1980)

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