The formation of [3H]inositol phosphates in human platelets by palmitoyl lysophosphatidic acid is blocked by indomethacin

Vol.

132,

October

No.

2, 1985

30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1985

THE FORMATION OF [3H]INOSITOL BY PALMITOYL LYSOPHOSPHATIDIC Stephen

P. Watson,

PHOSPHATES IN HUMAN PLATELETS ACID IS BLOCKED BY INDOMETHACIN

Marlene

Wolf

and Eduardo

Department of Molecular Biology, The Wellcome 3030 Cornwallis Road, Research Triangle Received

August

22,

555-562

G. Lapetina

Research Laboratories Park, NC 27709

1985

for platelet shape change SUMMARY. The intracellular Ca2+ thresholds aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoy;+lysophosphatidic acid involves an initial mobilization of intracellular Ca with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C. 0 1985 Academic Press, Inc. and

Lysophosphatidic and have

(l-6),

this

tissue

their

acids

therefore

effects

resembles

that

intracellular

platelet

activation

contrast

to Ca 2+ ionophores,

secondary)

Ca2+ by

of

the

however,

the

presence

of such

tion

response

is

observed

(4).

absence

oxygenase. requires

Abbreviation:

and The

results

the activation

of

inhibitors

show that

the

induction

of phospholipase

LPA16-palmitoyl

using acid

to

study

quin2) and

exert by the

required

A23187 only

(or

In

induce

the

full

inhibi-

primary)

(LPAlb)

for

(6).

cyclooxygenase

compares acid

a

within

acids

acids

a partial

lysophosphatidic indomethacin,

messengers

as demonstrated

exposed

The present

by palmitoyl presence

ionophores,

lysophosphatidic not

aggregation

lysophosphatidic

(estimated

in platelets

in

the

second

lysophosphatidic

(4);

activation

which

Ca2+

thresholds

tors

platelet

of human platelet

as possible

througti

decanoyl

aggregation

inducers

suggested

mechanism

similar

(or

potent

been

The

(3).

are

aggrega-

mechanism

of

and A23187

in

potent

inhibitor

of

of full

aggregation

cycloby LPA16

C.

lysophosphatidic

acid. 000&291X/85

555

$1.50

Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

132,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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METHODS Platelet preparation. Blood (200 ml) was obtained from drug-free volunteers (see 7). All experiments. with the exceotion of the auin2 fluorescence studies, were performed’ in a platelet’ aggregometer ‘with stirring. Platelet concentration was I.4 x 108/ml for stufes on platelets loaded with quin2, while a platelet concentration of 8 x 10 /ml was used for all other experiments. Labelin? with [32Pl and [3H]inositol. Platelets were resuspended in a modified Tyrode-HEPES buffer (134 mM NaCl, 12 mM NaHCOq, 2.9 mM KCl, 0.35 mM NaH2P04, 1 mM MgC12, 5 mM HEPES, 5 mM glucose, pH 7.4)-containing 1 mM EGTA. Prostacyclin (5 n5/ml) was added followed by either [32P]orthophosphate (5 mCi/lO ml) or [ Hlinositol (65-250 pCi/ml). Platelets were then left in a shaking water bath at 37°C for 1 h and 3 h, respectively. More prostacyclin (5 rig/ml) was added, They and the platelets were washed twice with buffer. were finally resuspended in EGTA-free buffer. Ca2+ (1 mM) was added 10 min before experimentation. [3H]Inositol phosphates were measured based on the method of Berridge et al. (8) as modified by Watson et al. (9); [32P] protein phosphorylation was estimated as described by Lapetina et al. (10). Loading with quin2. Platelets were loaded with quin2 in platelet rich plasma ( 1 1 \,+ separated as described above and resuspended in EGTA-free medium. Ca was added 10 min prior to experimentation. Quin2 fluorescence was measured as described by Rink et al. (11) and based on the method of Tsien et al. (12). Control experiments were performed in parallel on nonquin2-loaded platelets to enable corrections to be made for the fluorescence of A23187 and LPA16. Protein assayed for et al. (13).

kinase C assays. 5 min at 30°C with

Materials. and dissolved from Amersham sources (7,9,13).

Rat brain protein kinase C was purified and histone 1 as a substrate as described by Wolf

l-Palmitoyl-lysophosphatidic acid was purchased from Serdary tetraacetoxymethyl esters was in ethanol before use. Quin2, International. Other reagents were from previously described

RESULTS Shape shown)

Change

induced

The inclusion to

LPAI6

induced

either

(Figure

1B);

(0.1

which This

- 10 uM) was was

to

pattern

A23187

has

Phosphates.

relatively

been

termed

of blocked weak

all

the

(not

has been

termed

induced

three

[3H]inositol

in action;

the

presence maximally 556

full these or

primary a

(not

quin2

loaded

aggregation

(4). response

conditions, just

LPAI6

shape

change

aggregation

(4).

concentration-dependent phosphates

of

A23187

aggregation

shown)

LP'416

of

secondary

Under

1C).

1A) and

aggregation

blocked

(Figure

pattern

(Figure

full

aggregation

response

formation

LPAI6

by

however,

a reversible

completely a

Both

followed

indomethacin,

not

this

[3H]Inositol

change

response of

but

Aggregation.

shape

this

platelets;

and

indomethacin

effective

(Figure (not

concentrations

21,

shown). of

Vol.

132.

Figure

No. 2. 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

70 nM

303 Ilk4

5fxl IlM

1,ooa nM

30nM

50 nM

loo IlM

300 nM

COMMUNICATIONS

Shown are 1. Platelet Shape Change and Aggregation Responses. representative traces from one experiment, which is typical of at least three others. The traces in b and c are from the same A decrease in light transmission reflects the shape experiment. change of platelets from a disc to a spherical shape, and an increase in transmission reflects aggregation. a.

1-Palmitoyl-lysophosphatidic acid (LPAI6). Platelets were loaaea with qulnr In platelet ricn plasma and then separated by centrifugation in the absence of prostacyclin. They were resuspended in buffer to which 1 mM Ca2+ was added 10 min prior to addition of the lysophosphatidic acid.

b.

LPA16. Conditions lndomethacin (20

c.

uM)

were as during

Conditions were -.A23187 same experiment as b.

in a, except experimentation. as

in

b

for

and

the

the

trace

inclusion

is

from

of

the

10 63 Q x

6-

F 8

42O-

-----xr 1

I

0.01

0.1

CONCENTRATION

Figure

2.

Concentration Curve 1:Palmitoyl-Lysophosphatidic

for

the

I

I

1.0

10

LPA16

(IrM)

Formation of [3H]Inositol Phosphates by Acid (LPA16). Platelets labeled with ~‘HJlnOSltOl -(L5U Wl/mlI were exposea’“to various concentrations of LPAlb for 60 s and [3H]inositol phosphates determined. Each point is the mean + S.E.M. of triplicates from one experiment. This experiment is- representative of two others that yielded similar results. IP, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate. 557

Vol. 132, No. 2, 1985

induced

LPA16

BIOCHEMICAL

less

[3H]inositol

than

a

phosphates.

AND BIOPHYSICAL

100%

increase

Similar

in

results

RESEARCH COMMUNICATIONS

the

were

levels

of

observed

all

with

three

A23187

(not

shown). Relationship responses. by

between

Quin2

was used

concentrations

aggregation

I).

LPA16,

however, for

The

were

a

phosphorylated

the

selectively

kinase

Table

Agonist

I.

two

40,000 C (Figure

initiated

responses

for

there

maximal quin2

to

platelets

while

3). the

protein, A23187, 20,000

Thresholds Aggregation

A23187

and

that

the

the one for

maximal

A23187

responses.

C phosphorylates light

chain

a kinase

1,2-Didecanoylglycerol

the

reflecting

the

presence

protein

for the Induction of Shape in Quin2-loaded Platelets

Ca*+ threshold for shape change (nM)

agonists

Ca2+ concentrations

myosin

thereby

dalton

750 nM

two

possibility

kinase

(10).

in

the

than

of these Protein

dalton

the

and The

and

by

distinguish

studies.

protein

for

response

change

350 nM

mobilization was

produced

inhibitors.

were

Ca2+

estimation

dalton

shape

different

and

of

in

20,000

Ca2+

to cyclooxygenase

had a lower

an accurate

phosphorylated

exposed

curves

ability

Ca2+ levels

that

parallel;

protein

intracellular

significantly

phosphorvlation

dalton

protein

not

poor

1 UM prohibited

phosphorylates

of

not

concentration

The

Protein

not

the

and platelet

A23187

these

were

LPA16 may have

shown).

40,000

and

for

these

(Table

above

LPA16

thresholds

respectively;

(not

of

Ca2+ concentration

to estimate

of human platelets

estimated

curve

intracellular

of

at

only activation

exogenous

low

Change

concentrations

and

ca*+ for

threshold aggregation (nM)

LPA16

350

+

15

757

+ 94

A23187

362

+

13

758

c 98

Platelets were loaded with quin2 and suspended in ECTA-free Tyrode solution. Ca2+ (1 mM) was added to the platelets 10 min before the agonist. Initially, the threshold concentrations required to produce shape change and aggregation were determined in a platelet aggregometer; subsequently, the maximal quin2 fluorescence produced by these concentrations were measured in a fluorimeter. Each result is the mean + S.E.M. of four experiments. The resting Ca2+ level LPA16, palmitoyl-lysophosphatidic acid. was 108 + 3 nM.

558

Ca2+,

Vol. 132, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,,,

100 0.01

0.05

0.1

0.5

1.0

LOG CONCENTRATION

Figure

and

a.

40,000 dalton

protein

(40K).

b.

20,000 dalton

protein

(20K).

by

seen with consistent

of

Ca2+;

activated

A23187,

not

of

observation

had been partially

purified

Cross-desensitization not

containing (Figure

exposed no

4).

with

to

myosin

light

increasing

protein

that

This chain

levels

kinase

of

from rat

Ca2+,

Ca2+

responded doses

does not brain

and

of

to

(not

3).

activate

can

be

at

low

protein

LPA16

with

produced

resembled These

that

data

protein

were

kinase

shown).

prepared

subsequently

LPA16 559

that

(Figure

Platelets

prostacyclin

of

phosphorylation

LPAlb

studies.

EGTA or Addition

of

concentrations.

1,2-didecanoylglycerol

the

plasma

higher

(14).

a pattern but

at

activation

platelet

induced

with

proteins

selective

C was also LPA16

that

both

the

concentrations kinase

10.0

3. Protein Phosphorylation Studies in Human Platelets. Platelets labeled with [3CP]orthophosphate were exposed to various agonists for 60 s and protein phosphorylation determined. Each point is the mean + S;E.M. of triplicates from one experiment, and this experiment is representative of two others that yielded similar results. LPA16, l-palmitoyl-lysoDClo, 1,2-didecanoylglycerol; phosphatidic acid.

phosphorylated

explained

3.0 (IrM)

from resuspended reversible a

greatly

platelet in

rich medium

aggregation diminished

C

Vol.

132,

No. 2, 1985

Figure

4

(not

observed

by others

and

A23187

ments was

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

l-PalmitovlLysophosphatidic Reid and l-Oleoyl-2-Acetyl-Glycerol. Equivalent doses (in terms of response) of these three agonists were administered to platelets. When the aggregatory response had either leveled off or was returning back to control values a second dose of a different agonist was then administered. Shown are representative recordings from a single experiment. Similar results were observed in two other experiments.

response

Under

BIOCHEMICAL

shown).

Thus,

the

were

desensitized,

a phenomenon

(15).

these

conditions,

equivalent

cross-desensitized

were

performed

observed

with

was independent

platelets

with either

of the

doses

(Figure

4).

(in

terms

In contrast,

1,2-didecanoylglycerol, A23187

sequence

or

of

response)

when

similar

a synergistic

of the agonists

experi-

aggregation

l-palmitoyl-lysophosphatidic

of addition

of LPA16

acid (Figure

which

4).

DISCUSSION It phosphatidic platelets,

has

been

reported

acid

which

raise

the

that initiate

intracellular

concentrations shape Ca2+ 560

change levels

of

A23187

of

indomethacin-treated to

and

approximately

decanoyl

lysohuman

500 nM,

as

BIOCHEMICAL

Vol. 132, No. 2, 1985

indicated

with

quin2

cyclooxygenase

(6).

this

350 nM for

acid,

LPA16.

Further,

full

aggregation These

lipase

rather

the

induces

are

two sets

for

by monitoring

explanation A23187

that

and

is

to

In reach

inhibitors,

with

this

A2 with

acid.

The thromboxanes

lipase

C by acting

stable

endoperoxides

glycerol shown

which that

platelet for of

cyclooxygenase

interaction This

is

(Figure

inhibitors

between supported

synergize

by A23187

the

increased

by the

with

both

that

is

to raise

the the

formed

then

in C.

the

Kaibuchi

LPA16

the the

of

Ca2+

level

of

such

activation of

of

arachidonic

activate

et

phospho-

Ca2+

in of

formed

in

level

acids

had

(15)

A23187

result

with

of diacyl-

al.

with

newly

action

demonstrated

intracellular

likely

one

intracellular

liberation

lysophosphatidic

that

the

study

Thus,

initial

and metabolism

synergistic

Ca2+ and

and

the

as previously

lower

observation

A23187

phosphates.

about

results

most

present

brings

thus

and

is

made in the

is

A23187

cyclooxygenase

absence

are

the

of

In

kinase

of diacylglycerol

activation

produce

that

change.

receptors,

protein

therefore,

to

by phospho-

the

release

This

played

inhibitors,

endoperoxides

activates

doses

data

Ca2+

surface

role

[3H]inositol

shape

(7,8,18,19).

activation;

platelet

and

acids

absence

cyclooxygenase

subsequent

on cell

then

low

of

increased

the

the

on platelets

induce

the

of

lysophosphatidic

has been demonstrated

has been

compatible

acids

to the

in

of

500 nM to

of

lysophosphatidic

It

observation

presence

absence

decreased

species

related

levels

however

phospholipase

likely

the

the

in the

aggregation.

C activation

a similar

that

significantly

conditions

of conditions.

lysophosphatidic

Ca2+ levels. needs

most

is

and another

partial

RESEARCH COMMUNICATIONS

has shown

threshold

these

phospholipase (16,17);

study

A23187

than

inhibitors LPA16

both under

differences

C in

only

The present

inhibitors

approximately

AND BIOPHYSICAL

causing required

the

absence

a synergistic diacylglycerol.

1,2-didecanoylglycerol

is

to

activation

produce

platelet

able

to

4). Al though

A23187

platelet

activation

readily

distinguished

and through

Lysophosphatidic a

by the ability

similar

acids mechanism

of A23187 561

appear of

to produce

to

action, full

bring they

about can

aggregation

be of

Vol.

132.

No. 2, 1985

in the presence

platelets

this

BIOCHEMICAL

may

simply

be

intracellular

BIOPHYSICAL

of cyclooxygenase

that

A23187

Ca2+ level

AND

is

than that

able

RESEARCH

inhibitors. to

cause

COMMUNICATIONS

The explanation

a greater

for

increase

caused by lysophosphatidic

in

the

acids.

ACKNOWLEDGMENTS We are grateful to Dr. Pedro Cuatrecasas for invigorating discussions, Bryan Reep for excellent technical assistance, Randy McConnell for purifying I-decanoyl-lysophosphatidic acid and Lee Rose for expert preparation of this manuscript. REFERENCES 1. 2. 3.

Gerrard, J. M., Butler, A. M., Peterson, D. A., and White, J. G. (1978) Prostaglandins Med. 1, 387-396. Tokumura, A., Fukuzawa, K., Isobe, J., and Tsukatani, H. (1981) Biochem. Biophys. Res. Commun. 99, 391-398. Benton, A. M., Gerrard, J. M., Michiel, T., and Kindom, S. E. (1982) Blood

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60,

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MacIntyre, D. E., Shaw, A. M., Pollock, W. K., Marks, G., and Westwick, J. (1983) in Adv. Prostagland. Thromb. Leuk. Res. (Samuelsson, B., Paoletti, R. & Ramwell, P., eds.), Vol. II, pp. 423-428, Raven Press, New York. Simon, M.-F., Chap, H., and Douste-Blazy, L. (1984) FEBS Lett.

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5. P., McConnell,

Watson,

R. T.,

and

Lapetina,

E. G.

Biochem.

(1985)

J.

831-837..

Siess,

W., Siegel,

F., and Lapetina,

E. G. (1983)

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Chem.

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Berridge,

M., Downes,

C. P.,

and

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M.R.

(1982)

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J. Biol. Watson, S. P., McConnell, R. T., and Lapetina. E. I;. (1984) Chem. 259, 13199-13203. Lapetina, E. G., Watson, S. P., and Cuatrecasas, P. C. (1984) Proc. Natl. Acad. Sci. 81, 7431-7435. Rink, T. J., Smith, S. W., and Tsien, R. Y. (1982) FEBS Letts. 148,

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Wolf, M., Sahyoun, N., Levine H., III & Cuatrecasas, P. (1984) Biochem. Biophys. Res. Commun. 122, 1268-1275. Kaibuchi, K., Takai, Y., Sawamura, M., Hashijima, M., Fujikura, T., and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704. Hallam, T. J., Scrutton, M. C., and Wallis, R. B. (1983) FEBS Letts.

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P. (1981)

J. Biol.

Rittenhouse, S. E. (1984) Siess, W., Siegel, F. L.,

Biochem. J. 222, 103-110. and Lapetina, E. G. (1983)

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11236-11242. 19.

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Pollock, W. K., Armstrong, A. A., Brydon, MacIntyre, D. E. (1984) Biochem. J. 219,

562

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Jones,

R. L.,

and