monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 31, 1983

Pages

DIGLYCERIDE/MONOGLYCERIDE

LIPASES

PATHWAY IS NOT ESSENTIAL

ARACHIDONATE RELEASE IN THROMBIN-ACTIVATED Lee-Young Division

Received

April

Chau and Hsin-Hsiung

241-247

FOR

HUMAN PLATELETS Tail

of Medicinal Chemistry and Pharmacognosy College of Pharmacy University of Kentucky Lexington, Kentucky 40536-0053

19, 1983

Summary: Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation. Rapid thrombin

of phosphatidylinositol

stimulation

idonoyl is

turnover

is

diglyceride

thought

(1).

to be accompanied

Degradation

to be catalyzed Further

(2,3).

known

in human platelets

metabolism

of diglyceride the presence catalyzed

was proposed

as substrates, platelet

of a diglyceride

actually

lipase

are

pathway lacking.

lipase

and its

appears

should

in platelet of diglyceride

release

which

combines

C and a diglyc-

labeled

diglyceride

from diglyceride

by

the sequential

action

of a diglyceride

Diglyceride

lipase

catalyzes

and monoglyceride

lipase

2-arachidonyl monoglyceride this novel pathway combining

to be attractive,

significance

lipase

positional

of arachidonate

at sn-1,

We now demonstrate

1 To whom correspondence

mixed

in releasing the

transient

C

was shown by Bell

phospholipase

(5).

of the resulting (5,6). Although

C and two lipases

of this

release

of diglyceride

lyzes the hydrolysis donate and glycerol

to diglyceride

deacylation

arachidonate

Using

required

and a monoglyceride

the deacylation

lets

that

the

in arach-

phospholipase

to arachidonate

effectively

(4).

we found

membranes

lipase

rise

of phosphatidylinositol

Accordingly, a novel pathway for (4). the action of a phosphatidylinositol-specific lipase

by a transient

by a phosphatidylinositol-specific

--et al. who demonstrated membrane fraction which

eride

following

evidence

for

arachidonate accumulation

first

then

cata-

to arachiphosphothe

existence

in intact

plate-

of labeled

be addressed. 0006-291X/83

241

$1.50

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 113, No. 1, 1983 arachidonoyl

monoglyceride

prelabeled that

arachidonate

following

of a specific

abolishes

the

AND BIOPHYSICAL

in addition

human platelets

addition

platelets

BIOCHEMICAL

thrombin

inhibitor

transient

to labeled for

rise

RESEARCH COMMUNICATIONS arachidonoyl

stimulation.

diglyceride

of labeled

diglyceride We further

lipase monoglyceride

in show

to prelabeled but

not

release. MATERIALS 14

Materials: [lC]Arachidonic Human thrombin and essentially 1,2-Diglyceride and 2-monoolein tories, Inc., London, Ontario, hexanone oxime)hexane was kindly Group. Precoated silica gel G Chemist, Inc., Cincinnati, OH. from the Central Kentucky Blood

AND METHODS

acid (57.6 mCi/mmol) was purchased from Amersham. fatty acid free bovine albumin were from Sigma. were purchased from Serdary Research LaboraCanada. RHC-80267 (1,6-di(O-carbamoyl)cyclosupplied by Dr. C. Sutherland of Revlon Health plates were products of MCB Manufacturing Fresh human platelet concentrates were obtained Bank, Lexington, KY.

Preparation of 14 C-arachidonate labeled platelets: Fresh human platelet concentrates from one unit of blood were provided by the blood bank within 15 hr of withdrawal from normal donor. Platelet concentrates were centrifuged at 1000 xg for 10 min at 4'C. Platelets were resuspended gently in 25 ml of Triscitrate-bicarbonate buffer, pH 7.0, containing 10 mM citrate-dextrose and 1% fatty acid free bovine albumin (7) and then incubated at 37°C for one hour with 7.5 uCi [l-14C]arachidonate which had been bound to albumin (8). After centrifugation at 1000 xg for 7 min at 4"C, platelet pellet was washed once with the same buffer described above and finally resuspended in buffer without citrate and albumin to a concentration of 4 x log platelets/ml. Plastics and siliconized glassware were used throughout. Stimulation of platelets by thrombin: Labeled platelet suspension was warmed to room temperature before running experiments. For studying effect of RHC 80267 platelets were preincubated-with 10 uM RHC 80267 for 15 min prior to thrombin stimulation. Reaction was initiated by adding 8 units of thrombin (in 40 ul of deionized water) to 1 ml of platelet suspension which was kept stirring at room temperature. After incubation with thrombin for the indicated time, 3.75 ml of CHC13/MeOH (1:2) was poured into reaction mixture and platelet lipids were extracted as described by Cohen --et al. (9). For complete recovery of thromboxin B2 and prostaglandins, methanol-water nhase was acidified by adding 50 ul of acetic acid, and re-extracted twice with 2 ml of CHC13/MeOH (5:l). The lower CHC13 phase was combined and evaporated under nitrogen at 37°C. Chromatographic separation of lipid extracts: Lipid extract was fractionated by silicic acid column chromatography (10). Neutral lipid fraction was eluted by CHC13 and analyzed by two dimensional thin layer chromatography. Lipid samples with standards were applied to silica G plates (7 x 10 m). The plates were developed to 5 cm in the first solvent system of CHC13/MeOH/NH40H (95:2:1). After heating in an oven (60°C) for 15 min, plates were run in second dimension for 8 cm with the solvent system of petroleum ether/ethyl ether/acetic acid (60:40:1). Diglyceride, monoglyceride, 12-HETE and arachidonate were well separated. The lipids were localized by exposure to iodine vapor and scraped into scintillation vial for counting. Thromboxane B2 and other prostaglandins eluted by CHC13fMeOH (95:5) from silicic acid column were analyzed by thin layer chromatography using the solvent system of benzene/dioxane/ Prostaglandin standards were added during TLC for acetic acid (2O:lO:l). identification. Diglyceride was assayed

lipase and diglyceride as described previously

kinase (5). 242

Diglyceride lipase activity assays: For diglyceride kinase activity, the

BIOCHEMICAL

Vol. 113, No. 1, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

reaction mixture contained: 50 nM l-[14C]stearoyl &glyceride, 5 mM ATP, 10 mM MgC12, and 100 ng of platelet microsomal protein in a total volume of 0.5 ml of 50 mM HEPES buffer, pH 7.0. The reaction was initiated by adding microsomal protein and terminated by adding 0.5 ml of CHC13/MeOH/ 1 N HCl (1:2:0.5, v/v) followed by 0.1 ml of 1 N HCl and 0.5 ml of CHC13. The lower CHC13 layer was removed after centrifugation and the MeOH-H20 phase was extracted once more with 0.5 ml CHC13. The combined CHC13 layer was then dried under N2. Lipid residue was redissolved in CHC13/MeOH (1:2) and spotted on a silica gel plate (2 x 10 m) with phosphatidic acid as standard. After development in a solvent system of CHCl3/MeOH/acetic acid (90:10:12) for 7 cm, the phosphatidic acid region was localized by exposure to 12 vapor and scraped off the plate for counting. For studying inhibitory effect of RHC SO267 on both diglyceride lipase and kinase activities, RHC 80267 was firstly dissolved in dimethyl sulfoxide and then diluted 100 fold into the reaction mixture. One percent of DMSO showed no inhibitory effect on both enzymes. RESULTS AND DISCUSSION Rittenhouse-Simmons labeled bin

diglyceride (4,11,12).

metabolism

of this

acylation

of diglyceride

since

for

lyzed

is

intact rise

of

shown in Fig. with

those

described

monoglyceride

lowing

thrombin

ately

by other was also

found

following

stimulation Labeled

and 12-hydroxyeicosatetraenoic represented

approximately

at 2 minutes that

following

diglyceride

transient

labeled

acid

off

does metabolize arachidonoyl

of this the

(data

not

also shown).

to monoglyceride monoglyceride 243

(14,15). cata-

enzyme has been

function

of this

We have prelabeled thrombin.

A

was observed

as

was consistent [14C]arachi-

at 30 seconds

continued

at about

20% and 10% respectively stimulation

platelets

peaking

of arachidonate (12~HETE)

(11). functioning

Interestingly,

arachidonate

and leveled metabolites

found

was obviously

them with

(1,12).

to

acid

formation

to accumulate

Labeled

was not

at 15 seconds

of diglyceride

laboratories

stimulation.

same figure.

diglyceride

appear

stimulation

to be demonstrated.

course

Three

to phosphatidic

(4,5),

and stimulated

that

one is the

to 2-monoglyceride

fraction

[14C]arachidonate

The time

donoyl

the

has yet

suggests

does not

in activated

the existence

membrane

[14 Clarachidonoyl

1.

pathway

of diglyceride

Although

lipase.

with

This

thromlabora

platelets.

following

was shown to increase

platelets

human platelets

platelets (9).

following

triglyceride

of diglyceride

the deacylation

in human platelet

enzyme in

labeled

of

in several

The first

The acylation since

kinase

by a &glyceride

demonstrated

in activated

to triglyceride.

acid

pathway

transient

rapidly

platelets

rise

existence

have been described.

the phosphorylation

phosphatidic

confirmed

of diglyceride

in arachidonate-prelabeled one is

and transient

human platelets

nature occurs

by a diglyceride

The third

a rapid

was subsequently

diglyceride

in activated

The second catalyzed

observation

substance

routes

to increase

reported

The transient

metabolic be favorable

first

in arachidonate-prelabeled This

stimulation.

tories

(1)

to rise

2 minutes

fol-

immedi-

as shown in

such as thromboxane increased

steadily

of arachidonate These in intact

was presumably

results platelets. further

B2

and released suggest The metabolized

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

15 30

60

RESEARCH COMMUNICATIONS

120

Time, set Fig.

1:

Transient accumulations of diglyceride and monoglyceride in thrombinstimulated human platelets. [14C]Arachidonate labelled platelets (4 x log/ml) containing 30,000 cpm in PI were stimulated with thrombin (8 U/ml) for various times as indicated. Lipids were extracted and analyzed as described in Materials and Methods. O-O,

to labeled

arachidonate

be present platelet

diglyceride;

in platelets aggregation

hibited

by further If metabolism

and subsequently

04,

since

a very

(5,6).

This

be induced

addition

of aspirin

of arachidonoyl to arachidonate

of diglyceride

tidylinositol

through

diglyceride

only

reported

for

for (17),

fact

that

and in-

monoglyceride arachidonate,and

resynthesis inhibition

of phosphaof diglycer-

in disappearance of transient rise in monoglycarachidonate release. A specific inhibitor for

a potent

human platelet

shown to

by the

to arachidonoyl

cycle

were

(16).

in providing is

acid

monoglyceride

and indomethacin

to phosphatidate

platelet diglyceride lipase on phospholipases A2 andC. inhibitor

was supported

lipase will undoubtedly aid in assessing lipases pathway in arachidonate release.

(18)

lipase

by 1-arachidonoyl

phosphatidylinositol

arachidonic

monoglyceride

is essential

ide lipase alone should result eride and decrease in overall

and Amin

active

A-A,

presumption

could

metabolism

diglyceride monoglyceride

monoglyceride;

and selective

(I50 = 4 m) We have found diglyceride

role of diglyceridel Recently, Sutherland

inhibitor,

RHC 80267,

which showed little that this compound lipase

244

the

and inhibits

is

for

canine

inhibitory effect also a potent

diglyceride

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RHC80267, )IM Fig.

2:

Effect of activities Both lipase described RHC 80267 O-O,

kinase

only

of prelabeled

with

thrombin

platelets

showed

amount than

in the absence

labeled

was supported intact

unchanged eride

its

diglyceride

for

that

release

metabolism

of RHC 80267

arachidonate

properties

due to slight

by RHC 80267

of arachidonate suggests

that

in arachidonate entirely

and to phosphatidate of diglyceride

proposition

thromboxane

synthesis

compound

if

not

dichotomous

pathway,

release

Direct

deacylation

have been

proposed.

245

not

remained diglyc-

to triglyceride metabolism

of

occur-concurrently

and diglyceride

lipases

A2 has been recognized

(data

Apparently,

to diglyceride/monoglyceride

by phosphalipase

amount

diglyceride/monoglyceride

could

lipase

inhibition

This

release. that

the

total

metabolites

to phosphatidate we showed

since

of this and its

in

labeled

in the presence

constant. induced

not

In fact,

was probably

arachidonate

since

but

Interestingly,

higher

by the presence

Preincuba-

diglyceride 3.

was slightly

remained

of RHC 80267

essential

to monoglyceride

on kinetic In addition

lipids

finding

presence is not

based

metabolites

that

in the

diverted

and its

2.

by stimulation

stimulation,

synthetase

was inhibited

lipasespathway in the presence

This

in Fig.

followed

in labeled

following

of RHC 80267.

by the

The fact

rise

released

kinase

as shown

as shown in Fig.

or thromboxane

platelets

shown).

a transient

progressively

arachidonate

diglyceride

10 uM of RHC 80267

was observed arachidonate

of cyclooxygenase

e-0,

concentrations with

that

increased

of labeled

lipase;

high

monoglyceride

arachidonate

in

diglyceride

at relatively

tion labeled

RHC 80267 on diglyceride lipase and diglyceride kinase from human platelet microsomes. and kinase activities were assayed under conditions in Materials and Methods with various concentrations of as indicated.

as a favorable

kinase other

(6).

pathways

of phosphopathyay

since

of

Vol. 113, No. 1, 1983

BIOCHEMICAL

P.

01530 Fig.

this Billah ide

3:

.

AND BIOPHYSICAL

60 Time,sec

of RHC 80267 on the formation of diglyceride and monoglycerthrombin-stimulated human platelets. platelets were preincubated with 10 PM of RHC 80267 for 15 to thrombin stimulation. Conditions of stimulation and of analysis are the same as indicated in Fig. 1.

O-O,

diglyceride;

the

simplest

m-4,

monoglyceride;

mode of release

a modified --et al. (20) proposed derived from phosphatidylinositol

donate

in activated catalyzed

lated

horse

platelets

phospholipids indicates that in arachidonate

which

initiated

entirely

by this

A2.

by phosphatidate

formed

diglyceride/monoglyceride lipases release, it does not necessarily

arachidonate

release

following

thrombin

be determined. 246

stimulation

in stimu-

deacylation

Although

pathway support

or modified phospholipase A2 pathways play a major role lease. The percent contribution by diglyceridelmonoglyceride overall

found

by direct

(22).

arachi-

The resultant phospholipids

Lysophospholipids and/or

diglycer-

to release

phospholipase

manner

However,

that

to phosphatidate,

by arachidonate-containing (21).

acid

(8,19).

A2 pathway

was effected

specific

process

can be derived

arachidonic

of arachidonate

was converted

platelets,

can be reacylated

transesterification

A-A,

phospholipase

by a phosphatidate

lysophosphatidate through

I

120

Effect ide in Labeled min prior methods

represents

at least

RESEARCH COMMUNICATIONS

our

of

finding

may not be essential phospholipase A2 and/ in arachidonate pathway still

reto

remains

to

Vol. 113, No. 1, 1983 Just Prescott tion

prior

BIOCHEMICAL

to submission

and Majerus

also

AND BIOPHYSICAL

of this

paper

demonstrated

for

very

of an arachidonoyl-monoglyceride

RESEARCH COMMUNICATIONS

publication,

recently

intermediate

the

we noted transient

during

that

accumula-

thrombin

stimulation

(23). ACKNOWLEDGEMENTS We are gift

indebted

of RHC 80267,

of the manuscript. Association

(81-1152)

(GM-30380,

GM-31454),

to Dr.

C. Sutherland

of Revlon

and to Ms. L. Benton This

work

and its

for

her

was supported Kentucky

and Kentucky

assistance

by grants

Affiliate,

Tobacco

Health

Research

from

National Board

Group

for

in the

his

preparation

the American Institutes

kind Heart

of Health

(4B-506).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Rittenhouse-Simmons, S. (1979) .I. Clin. Invest. 63:580-587. L. (1979) EBS Lett. 100:367-370. Mauco, G., Chap, H. and Douste-Blazy, Chau, L. Y. and Tai, H. H. (1982) Biochim. Biophys. Acta __ 71=44-351. Bell, R. L., Kennerly, D. A., Stanford, N. and Majerus, P. W. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3238-3241. Chau, L. Y. and Tai, H. H. (1981) Biochem. Biophys. Res. Commun. __ 100: 1688-1695. Chau, L. Y. and Tai, H. H. (1983) submitted for publication. Rittenhouse-Simmons, S. and Deykin, D. (1976) Biochim. Biophys, Acta 426:688-696. Rittenhouse-Simmons, S., Russell, F. A. and Deykin, D. (1976) Biochem. Biophys. Res. Commun. 70:295-301. Cohen, P., Broekman, MrJ., Verkley, A., Lisman, T. W. W. and Derksen, A. (1971) J. Clin. Invest. 50:762-772. Bills, T. K., Smith, B. andSilver, M. J. (1976) Biochim. Biophys. Acta 424:303-314. Homa, S. T., Conroy, D. M. and Smith., A. D. (1980) Biochem. Biophys. Res. Common. 95:1321-1327. Imai, A., Yanz K., Kameyama, Y. and Nozawa, Y. (1982) J. Exp. Med. (Japan) 52:99-105. Call, F.1. and Rubert, M. (1973) J. Lipid Res. 14:466-474. Lapetina, E. G. and Cuatrecasas, P. (1979) Biochim. Biophys. Acta --573: 394-402. Broekman, M. J., Ward, J. W. and Marcus, A. J. (1980) J. Clin. Invest. 66:275-283. crrard, J. M. and Graff, G. (1980) Prostaglandin Med. 4:419-430. Broekman, M. J., Ward, J. M, and Marcus, A. J. (1981) 3, Biol. Chem. -*256. 8271-8274. Sutherland, C. A. and Amin, D. (1982) J. Biol. Chem. --257:14006-14010. McKean, M. L., Smith, J. B. and Silver, M. J. (1981) J; Biol. Chem. 256: 1522-1524. Billah, M. M., Lapetina, E. G. and Cuatrecasas, P. (1981) J. Biol. Chem. 256:5399-5403. Lapetina, E. G., Billah, M. M. and Cuatrecasas, P. (1981) Nature __ 292: 367-369. Lapetina, E. G., Billah, M. M. and Cuatrecasas, P. (1981) 3. Biol. Chem. -256:5037-5040. Prescott, S. M. and Majerus, P. W. (1983) J. Biol. Chem. --258:764-769.

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