Vol. 90, No. 2, 1979 September 27, 1979

AND BIOPHYSICAL

BIOCHEMICAL

CALMODULIN STIMULATES Patrick

HUMAN PLATELET

Y.-K.

PHOSPHOLIPASE A2

Wang+ and Wai Yiu

Departments of Pharmacology+ The University of Tennessee Center Memphis, Tennessee

RESEARCH COMvtUNlCATlONS Pages 473-480

Cheung+

and Biochemistry+ for The Health Sciences 38163

and Department of Biochemistry+ Jude Children’s Research Hospital Memphis, Tennessee 38101

St.

Received

August

6,1979 SUMMARY

Ca 2+ -binding protein, mediating the effect of Calmodulin is a ubiquitous Ca2+ on many enzyme systems and cellular reactions. Phospholipase A2 (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) yhich governs the level of arachidonic acid in human platelets, requires Ca2 for maximum activity. Resylts presented herein suggest that the stimulation of phospholipase A2 by Ca2 is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin. INTRODUCTION Phospholipase lyzes

the

fatty

acid

and

(1).

The

steady

low

(2)

step

in

A2 (phosphatide-2-acyl-hydrolase,

deacylation state

generation

figure

Pickett, increased that

al

(4)

markedly

platelet

of this

fatty

The

the

level A, (5,

by which

free

stimulated

which

cata-

to produce all

mammalian

arachidonic

acid

believed

is

to be the

Arachidonic

acid

a free tissues

generally limiting

serves

and prostaglandins,

as a

compounds

Using

ionophore

A23187,

of

intracellular

Ca2

mobilization

of

is 6)

arachidonic by

acid,

+

indicating

intracellular

Ca2+.

investigated the metal requirements of + that Ca2 was required for maximal enzyme

A2 and showed

mechanism

(3).

in

aggregation.

that

phospholipase &

A, is

acid

platelet

platelet

g

of

thromboxanes,

demonstrated the

phospholipase

activity. been

phospholipase

in

present

level

of endoperoxides,

Rittenhouse-Simmons, platelet

is

of

importantly

et -2

of p4osphoglycerides

intracellular

activity

common precursor that

2 position

a lysophosphoglyceride,

and the the

at the

EC 3.1.1.4),

Ca2’

stimulates

phospholipase

A2 has

not

clarified.

+

Current

t Address

address:

correspondence

Department of Pharmacology New York Medical College-Valhalla, New York 10595 to this

author. 0006-291X/79/180473-08$01.00/0 473

Copyright AU rights

@I 1979 by Academic Press, Inc. of reproduction in any form reserved

Vol. 90, No. 2, 1979

Recent was

BIOCHEMICAL

evidence

first

from

discovered

esterase

is Calmodulin

and cellular

reactions, (11,

muscle

and

sarcoplasmic

the

disassembly

Ca2+ in

12),

A, is

important

to

ated

calmodulin.

indeed

brain

reticulum

pholipase

suggesting

including myosin

chain (18),

dependent

that

mediated

the

the

for

(13-16),

lo),

erythrocyte

NAD kinase

(17),

in erythrocytes

(19)

(21,

22),

and

calmodulin

mediates

the

effect

of

of

that

platelet

activity

(5,

membrane 6))

Ca2+ on phospholipase we

present

human platelet

preliminary

phospholipase

it

phos-

was deemed A2 is

medi-

evidence As by Ca 2+

is

calmodulin. AND METHODS

and Reagents

(l-14C)-2-arachidony1 phosphatidylcholine (55 from Applied Sciences; thin-layer chromatography Instruments; (l-14C)-arachidonic acid (56 mCi/mmole) and arachidonic acid from Nuchek Co. Prostaglandins gifts from Dr. U. Axen of Upjohn Co. Preparation

(9,

in

of enzymes

membranes

finding of

report,

MATERIALS Chemicals

ubiquitous

of

maximum

effect

this

stimulation

through

kinase

Ca 2t transport

that

and the

on Gas ’ In

cyclase

which

phosphodi-

Ca2+ on a variety

phosphorylation

evidence

whether

calmodulin,

(23).

reactions,

examine

of

adenylate

light

that

cyclic 3’,5’-nucleotide + protein Ca2 -binding

effects

kinase (20),

increasing

many cellular

by

the

phosphorylase

of the

of

mediates

of microtubules

In view

indicates

a multifunctional

(7, 81,

Ca2+ -ATPase

many laboratories

as an activator

eukaryotes.

skeletal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Human Platelet

mCi/mmole) was purchased plates from Brinkman from New England Nuclear; and thromboxane B2 were

Membranes

Newly expired platelets, collected by centrifugation at 700 x g for 10 min. to remove red blood cells, were sonicated for approximately 20 min. The homogenate was centrifuged at 7,000 x g for 20 min., and the supernatant fluid was centrifuged further at 105,000 x g for 60 min. The sediment was suspended in 50 mN Tris-HCl (pH 8.8) to make a protein concentration about 10 mg/ml. The sample was used fresh or stored at -20’. One unit of human platelets yielded approximately 2 mg of membrane protein. Assay

of Phospholipase

A2 in Platelet

Membranes

Each assay mixture (final volume 1 ml) contained 50 muCi of (l-14C)-2arachidonyl phosphatidylcholine, p$atelet membranes (approximately 1 mg), 50 mM Tris-HCl (pH 8.8), 0.1 mM Ca2 and 1 ull calmodulin. The substrate was dried under N2 and sonicated in 20 ul of ethylether to produce an emulsion. The reaction was started by the addition of platelet membranes. After incubation at 37O for 5 min. with constant shaking, the reaction was terminated by the addition of 0.2 ml of ice-cold 0.1 M HCl and 10 ug of arachidonic acid. Carrier arachidonic acid was added to facilitate the isolation of (l-14C)-arachidonic acid. The reaction mixture was extracted 3 times with 2 ml of n-hexane. The organic phase was separated from the aqueous phase by centrifugation at 5,000 x g for 5 min. After phase separation, the tube was

474

BIOCHEMICAL

Vol. 90, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chilled in a methanol-dry ice bath. The hexane phase was removed from the frozen aqueous phase with a Pasteur pipette, and was ttansferred to a scintillation vial. The extract was evaporated to dryness under a stream of air. The dry lipid residue was dissolved in 5 ml of a Triton X-100, toluene-based scintillation fluid for counting. Measurement

of Thromboxane

B2 Released

from

Washed Human Platelets

Platelet-rich plasma was passed through a Sepharose-2B column (1.5 x 20 cm) which had been equilibrated with Ca2 -free Tyrode buffer, pH 1.4 (24). The platelets emerged in the void volume of the column, and were well separated from plasma proteins. A fraction of the platelet suspension (0.5 ml) was incubated with (l-14C)-arachidonic acid (2 ug, 0.36 uCi) for IO min. at 37” with constant stirring in a Payton dual channel aggregometer in the presence or absence of 1 uM calmodulin. After aggregation of the platelets, as monitored by a decrease in light transmission, the reaction was terminated by acidification to pH 3.0 with 0.1 M HCl, and the suspension was extracted 3 times with 2 ml of ethylacetate. The organic phase was pooled and dried under N and the reaction product was separated by thin-layer chromatography, using a*iolvent system containing ethylacetate:acetic acid (99:1, v/v) (25). The reaction product was identified with an authentic thromboxane B, on the same plate (Rf value 0.57). This solvent system clearly separates thromboxane B, The radioactive peaks were localfrom PGE2, PGF2 , and 6-keto PGFi (25). ized with a Pack%rd 2730 radiochro%atogram scanner (Packard Instrument Co.). Preparation

and Assay

of Calmodulin

Calmodulin was purified from bovine brain (26, 27). The assayed by its ability to stimulate bovine brain calmodulin-deficient diesterase (26). The use of bovine brain calmodulin is warranted lacks both tissue and species specificity (28). Protein

protein was phosphobecause it

Determination

Protein was determined as a standard.

according

to Lowry

(29)

using

bovine

serum

albumin

RESULTS AND DISCUSSION Human platelets preparations lipase

A2 showed

protein.

When

arachindonyl only

one

a range these

radioactive acid

on

the

presence

In 1). two-fold,

the

presence

acid

(5,

of

platelet

in

accord

in

the

presence

the

co-migrated

the

previous

a plateau amount

findings

of exogenous increased, of EGTA.

per

with mixture

with The

release in

that

Ca 2+

calmodulin,

about acid

stimulates the

to

(l-14C)-2generated

an authentic

level

level

lOO-200%

small

Four

mg of membrane

reaction

arachidonic

The relatively

(30).

of phospho-

incubated

of

equivalent

475

calmodulin as a source

calmodulin

were

5 min.,

of

used

chromatography.

Ca*+,

In the presence 6). released was further

obtained

which

of EGTA reached with

were

membranes for

thin-layer of

amount

which

140 to 675 ng of

product

in

A2

a considerable membranes

phosphatidylcholine

arachidonic acid

contain

of the platelet

increase

sample

of

of

arachidonic

20 min. was

(Fig.

increased

phospholipase of arachidonic above

the

level

by exogenous

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

1

20

30

40

50

60

TME

(mid

Fig. 1 - Effect of calmodulin and Ca2+ on phospholipase platelet membranes. Fifty muCi of (l- r4C)-2-arachidonyl was dried in a test tube under N,, and sonicated in produce an emulsion. To this tube was added 50 mM Tris of platelet membranes in a final volume of 1 ml. After constant shaking for the times indicated, the amount released was determined as described under Materials lip$se A2 activity was expressed as arachidonic acid Ca2 , 0.1 mM; and calmodulin, 1 uM. calmodulin

is

presumably

phenomenon

reminiscent

erythrocyte

plasma

these apparent

leveling-off of

contain

reaction

from

and the The

Thus,

reacylation

known

mM.

phospholipase adenylate

represents

to

phospholipase of

to block

A2 by

Ca2+ an

Ca2+,

the

Ka being (32).

the

platelet

membrane

Trifluoperazine At; activity

cyclase

however, to (33)

preparations,

and EGTA had basal and

they

both

level. cyclic

effectively

nucleotide

476

of acid

the

release

was

further

in

phenothiazine

activity

of

calmodulin

to calmodulin

to

1, calmodulin

the

Similar

reacylation

calmodulin

bound

no effect

few tissues

2).

is bound

in Table

The

initial

between

biological

Calmodulin

As shown

the

antidepressive

trifluoperazine 10’.

and

both

calmodulin.

the

Fig.

and of

small;

of arachidonic

equilibrium (see

(9)

Mammalian

level

a

stimulation

after

catalyzes the

an

specifically

of also

reacylation.

trifluoperazine,

of

acid

membranes,

cyclase

extent was

acid

PhosphoEGTA, 1 mM;

the

endogenous

A2; the platelet membranes contained + Ca2 . Atomic absorption spectrophotometric

in 6 different

stimulated

of use

inactive

endogenous 0.8

due

to phosphoglycerides

presence

affinity,

biologically

to

which

the

was

probably

of

of arachidonic

acyltransferase,

the

phospholipase

release

in

adenyiate

The

levels

to phosphoglycerides.

by In

the

high

arachidonic

released.

calmodulin

active mixture

derivative high

in

(31).

exogenous

relatively

activation

examined

(32).

Ca2+-ATPase by

on brain

probably

acid

the

findings

enzymes

of

and Methods.

calmodulin

incubation

an

arachidonic

endogenous

membrane

retained

minutes

to

of previous

particulate

preparations

due

Aa activity.in human phosphatidylcholine 20 ul of ethylether to buffer (pH 8.8), 1 mg incubation at 37O with

with

trifluoperazine

is

itself

relatively

a

activated

high

levels

of

measurement showed that Ca2 + levels ranged from 0.4 on the suppressed

observations phosphodiesterase

basal the have

activity

of

calmodulinbeen (32).

made on These

BIOCHEMICAL

Vol. 90, No. 2, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Phospholipids Phospholipase Ca2

Acyltransferase

A,

II Arachidonic Acid

1

Cycle-oxygenase

Endoperoxides

Thromboxane Synthase I Thromboxane

A,

1 Thromboxane

B2

Fig. 2 - A simplified scheme showing the metabolism of arachidonic acid in human platelets. The level of arachidonic acid, which serves as a precursor is governed by its release from and of endoperoxides and thromboxanes, reacylation to phosphoglycerides. Platelet membranes do not further metabolize arachidonic acid. Whole platelets, however, convert arachidonic acid to the endoperoxides and thromboxane A,. Thromboxane A2 is unstable, and is readily hydrated nonenzymatically to form the more stable thromboxane B,.

TABLE EFFECT

OF Ca2+

OF PHOSPHOLIPASE

1

AND CALMODULIN A,

ON THE ACTIVITY

IN HUMAN PLATELET

MEMBRANES

Additions

Activity (f

mol/mg/5

None

18.8

EGTA

21.1

Trifluoperazine

18.0

Ca2+

42.3

Calmodulin

min.)

38.0

Calmodulin,

Ca2+

47.9

Calmodulin,

EGTA

18.9

Calmodulin,

Ca2+,

Trifluoperazine

23.6

Each reaction mixture in a final volume of 1 ml contained 50 muCi of (1_14C)2-arachidonyl phosphatidylcholine, 50 mM Tris-HCl (pH+8.8), approximately 1 mg and where present 0.1 mM Ca2 , 1 uM calmodulin, 1 mM of platelet membranes, EGTA, or 0.15 mM trifluoperazine. TDifluoperazine was dissolved in 50 mM Tris-HCl (pH 8.8). The reaction mixture was incubated for 5 min. at 37O with constant shaking. Phospholipase A2 activity is expressed as fmoles of arachidonic acid released per mg protein for 5 min.

477

Vol. 90, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 3 - Increased formation of thromboxane B, by calmodulin in human platelets. Washed platelets (0.5 ml) were incubated with fl-r4C)-arachidonic acid (2 ug, 0.36 uCi) at 37OC in the presence (A) or absence (B) of 1 uM calmodulin, with constant stirring (1,200 rpm) in an aggregometer for 10 min. At the end of the incubation, the reaction product was extracted and separated by thin-layer chromatography as described in Materials and Methods. The radioactivity on the thin-layer plates was scanned by a Packard 2730 radiochromatogram scanner. The’zone corresponding to TXB, was scrapped and extracted with methanol. The extract was dried and the radioactivity was determined in a liquid scintillation counter. Tliromboxane B, zone in Panel A gave 129,360 dpm, that in Panel B 58,560 dpm.

results

support

mediated

the notion

through human

exogenous

with

fication,

and the

donic boxane B,

boxane

One other

Ba from The

05).

lipids

(34). B,

of

solvent

(Fig.

for

products

A majority

The

on phospholipase

the

A, is

used

formed

the was

After the

isolated

in

and 6-keto

increased

of

reaction

was terminated

separated

identified

this

experiment

PGF1o, at

of exogenous approximately

478

from

acid

was

by acidichroma-

as unchanged

arachi-

as thromboxane clearly

which

the

B,

by thin-layer

was recovered

spot,

using

thromboxane

(l- i4C)-arachidonic

and

remaining

presence

was corroborated

formation

radioactivity

radioactivity In

3).

radioactive PGF,o

the

10 min.,

were

system

PGEa,

A, by calmodulin

measuring

acid

platelets

acid.

noted.

by

arachidonic

.

of Ca 2+

stimulation

of phospholipase

platelets

incubated tography

the

calmodulin.

The stimulation washed

that

migrate

origin

is

calmodulin, two-fold.

B,,

separates behind presumably

the

was

throm-

thromboxane phospho-

amount

of

The

effect

thromof

BIOCHEMICAL

Vol. 90, No. 2, 1979

exogenous intact

calmodulin

platelet

on

is

exterior

surface

let

and acts

on the

of

phospholipase added

and

the

thromboxane

of the

synthase (see

The

increase

of

effect

mediated

tion.

2).

of Cazt like

finding

that

The

of

effect

cellular

synthesis

B,

from arachidonic

plateagonist

The arachidonic

thromboxane

product,

A, and

from is

thromboxane supports

A, has been Results

numerous

enzymes

stimulates

noted

the for

presented

mentioned platelet

enzymes

the

rapidly B,

(36).

notion

that

the

provides

a molecular

and

plays

in

suggest

link

between role

in

that

regula-

interwoven

CAMP and the

is

A, adds to

cellular are

of

its

INTRODUCTION,

phospholipase involved

metabolism

a pivotal

some time,

herein under

of CAMP and prostaglandins

controlling thus

the

on

by cycle-oxygenase t Ca2 for activity.

short-lived acid

the

A2 in human platelets.

and metabolism

regulators,

(35).

requires

is

stable

elucidated.

In

in

effect

into

utilized of

A,

a more

its

transported

them

to

calmodulin

(37). calmodulin

glandins,

the

calmodulin-regulated

instances

exerts

platelets

of

Thromboxane

the

calmodulin.

list

intact

B,

a known physiological

to be readily

on phospholipase

has not been

or is

neither

catalyzes

through

The

many

(34),

phospholipase

of Ca2 ',

growing

on the

thromboxane

either

Thrombin,

appears

thromboxane

effect

mode of action

the

acts

Fig.

regulates

The the

also

non-enzymatically

calmodulin

membrane,

RESEARCH COMMUNICATIONS

of

calmodulin

surface.

synthase

endoperoxides converted

plasma

platelets

Thromboxane

formation

interest;

interior

A,,

to

increased

of special

the

acid

the

AND BIOPHYSICAL

two

in

prosta-

classes

cellular

of

regulation.

ACKNOWLEDGMENTS We are Children's

grateful Research

T. K. Narayanan photometry; work

was

(PY-KW), is

for

to

Hospital

Skip for

Skinner

grants

supplies

of calcium

W. H. Lee for

by

and Mr.

generous

the determination

and to Mr. supported

Mr.

preparation

NS 08059

of a Young

Investigator

Award

Kunkel

Heart

of

of human platelets; by atomic

absorption

of platelet

(WYC),

by ALSAC (WYC) and by the American

a recipient

Keith

CA 21765

microsomes. (WYC),

Association

St.

Jude to Dr.

spectroThis

and DE 40591

(PY-KW).

PY-KW

(HL 22075).

REFERENCES 1.

2. 3. 4.

Vogt, W. T., Suzuki, T., and Babilli, S. (1966) Memoirs of the Society for Endocrinology, pp. 137-142, University Press, Cambridge. Marcus, A. J., Ullman, H. L., and Safier, L. B. (1969) J. Lipid Res. 10, 108-114. Bergstrom, S., Danielsson, H., Klemberg, D., and Samuelsson, B. (1964) J. Biol. Chem. 239, 4006-4008. Pickett, W. C., Jesse, R. L., and Cohen, P. (1977) Biochim. Biophys. Acta 486, 209-213.

479

Vol. 90, No. 2, 1979

5. 6. ii: 9. 10.

11. 12. 13. 14. 15. -16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. E: 34. 35. 36. 37.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Rittenhouse-Simmons, S., Russell, F. A., and Deykin, D. (1977) Biochim. Biophys. Acta 488, 370-380. Rittenhouse-Simmons, S., and Deykin, D. (1978) Biochim. Biophys. Acta 543, 409-422. Cheung, W. Y. (1967) Biochem. Biophys. Res. Commun.29, 478-482. Cheung, W. Y. (1970) Biochem. Biophys. Res. Commun.38, 533-538. Cheung, W. Y., Bradham, L. S., Lynch, T. J., Lin, Y. M., and Tallant, E. A. (1975) Biochem. Biophys. Res. Commun.66, 1055-1062. Brostrom, C. O., Huang, Y. C., Breckenridge, B. M., and Wolff, D. J. (1975) Proc. Natl. Acad. Sci. USA 72, 64-68. Gopinath, R. M., and Vincenzi, F. F. (1977) Biochem. Biophys. Res. Commun.77, 1203-1209. Jarrett, H. W., and Penniston, J. T. (1977) Biochem. Biophys. Res. Commun.77, 1210-1216. Dabrowska, R., Sherry, J. M. F., Aromatorio, D. K., and Hartshorne, D. 3. (1978) Biochemistry 17, 253-258. Waisman, D. M., Singh, T. J., and Wang, J. H. (1978) J. Biol. Chem. 253, 3387-3390. Yagi, K., Yazawa, M., Kakiuchi, S., and Ohshima, M. (1978) J. Biol. Chem. 253, 1388-1340. Hathaway, D. R., and Adelstein, R. S. (1979) Proc. Natl. Acad. Sci. USA 76, 1653-1657. Anderson, J. M., and Cormier, M. J. (1978) Biochem. Biophys. Res. Commun. 84, 595-602. Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, T. C., and Nairn, A. C. (1978) FEBS Lett. 92, 287-293. Hindo, T. R., Larsen, F. L., and Vincenzi, F. F. (1978) Biochem. Biophys. Res. Commun.81, 455-461. Katz, S., and Remtulla, M. A. (1978) Biochem. Biophys. Res. Commun.83, 1373-1379. Schulman, H., and Greengard, P. (1978) Nature 271, 478-479. Schulman, H., and Greengard, P. (1978) Proc. Natl. Acad. Sci. USA 75, 5432-5436. Narcum, J. M., Dedman, J. R., Brinkley, B. R., and Means, A. R. (1978) Proc. Natl. Acad. Sci. USA 75, 3771-3775. Tangen, O., Berman, H. J., and Marfey, R. (1971) Thromb. Diath. Haemorrh. 25, 269-278. Gorman, R. R., Bundy, G. L., Peterson, D. C., Sun, F. F., Miller, 0. V., and Fitzpatrick, F. A. (1977) Proc. Natl. Acad. Sci. USA 74, 4007-4011. Lin, Y. M., Liu, Y. P., and Cheung, W. Y. (1974) J. Biol. Chem. 249, 4943-4954. Wallace, R. W., and Cheung, W. Y. (1979) 3. Biol. Chem. (in press). Cheung, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Smoake, J. L., Song, S.-Y., and Cheung, W. Y. (1974) Biochim. Biophys. Acta 341, 402-411. Lynch, T. J., and Cheung, W. Y. (1979) Arch. Biochem. Biophys. 194, 165-170. Levin, R. M., and Weiss, B. (1976) Mol. Pharmacol. 12, 581-589. Brostrom, M. A., Brostrom, C. O., and Wolff, D. J. (1978) Arch. Biochem. Biophys. 191, 341-350. Minkes, M. N., Stanford, N., Chi, M., Roth, G. J., Raz, A., Needleman, P and Majerus, P. W. (1977) J. Clin. Invest. 59, 449-454. B&s, T. K., Smith, J. B., and Silver, M. J. (1977) J. Clin. Invest. 60, l-6. Hamberg, M., Svensson, J., and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. USA 72, 2994-2998. Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Hammarstrom, S., and Molmsten, C. (1978) Ann. Rev. Biochem. 997-1029. 480

Calmodulin stimulates human platelet phospholipase A2.

Vol. 90, No. 2, 1979 September 27, 1979 AND BIOPHYSICAL BIOCHEMICAL CALMODULIN STIMULATES Patrick HUMAN PLATELET Y.-K. PHOSPHOLIPASE A2 Wang+ a...
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