Effects on adenylate cyclase activities of unsaturated fatty acid incorporation into rat liver plasma membrane phospholipids specific modulation by linoleate

Vol. July

95. No.

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

16. 1980

97-102

EFFECTS ON ADENYLATE CYCLASE ACTIVITIES OF UNSATURATED FATTY ACID INCORPORATION INTO RAT LIVER PLASMA MEMBRANE PHOSPHOLIPIDS. SPECIFIC MODULATION BY LINOLEATE. Odile

Colard,

Aline

Kervabon

and Christian

Roy*

Laboratoire de Biochimie, C.H.U. Saint-Antoine, 27, rue Chaligny, 75571 Paris Cedex 12, France and*Laboratoire de Physiologie cellulaire, College de France, 11 place Marcelin-Berthelot, 75231 Paris Cedex 05, France.

Received

February

14,

1980

SUMMARY The adenylate cyclase activities of the rat liver plasma membrane were measured simultaneously with the incorporation of acyl chains into the membrane phospholipids using oleyl CoA, linoleyl CoA or arachidonyl CoA thioester. The basal, fluoride - and glucagon - stimulated adenylate cyclase activities were increased by the incorporation of linoleate into the plasma membrane phospholipids. Oleyl CoA did not alter the adenylate cyclase activities whereas arachidonyl CoA, at high concentration, decreased the adenylate cyclase activities. These data indicate a specific effect of phospholipid molecular species containing linoleate. INTRODUCTION Several importance

experimental of membrane

example,

membranes

solvants

(4)

ments Houslay

and al in both

cyclase

; Engelhard

and PGEI-

have

(6)

been treated

fused

the

and al

polar

(9)

in rat

activity,

in LM cell

membranes

and fatty

reticulocytes

These

membranes

modifications acid

cyclase

or both. and observed

of adenylate

as a result

fluorideof the

compositions

to the

phosphatidylethanolamine

treat-

of basal,

have correlated

For

2, 3) organic

and the activity

8) reported

adenylate into

(1, stimulation

with

the

activities. (5).

hormone

vesicles

headgroup

the isoproterenol-sensitive phosphatidylcholine

cyclase

phospholipases

dependancy

(7,

activities

of the

with

phospholipid

and al

adenylate

to evaluate

and lysophospholipids

in basal

temperature

stimulated

manipulation

for

detergents

in changes

changes

have been employed

phospholipids

non ionic

resulted

Axelrod

approaches

; recently

the activation

of

transmethylation

and to changes

of in membrane

fluidity. Lysophosphatidylcholines adenylate stimulated rat

liver

cyclase

activity

adenylate plasma

have been demonstrated cyclase

membranes

as well

as fluoride

activities.

to inhibit (5,

11) or glucagon

Lysophospholipids

and can be produced

basal

by the

are

(lo) (11)

present

phospholipase

in

A acti-

0006-291X/80/130097-06$01.00/0 97

Copyright Z, 1980 by Academic Press, Inc. A II rights of reproduciron in an-v ,form reserved.

Vol.

95, No.

1, 1980

BIOCHEMICAL

vities

(12,

13,

porate

long

chain

Such

transacylation

decrease

in

cular

14).

It

was

fatty

reported

acyl-CoA could

lysophospholipid which

evaluate

the

RESEARCH

that

the

membranes

modify

plasma in

represent

amount

would

BIOPHYSICAL

thioesters

processes

species

AND

the

lipid

could

incor-

phospholipids

a mechanism

and/or the

their

COMMUNICATIONS

allowing

formation

of

environment

(15).

of

the

the

specific

mole-

adenylate

cyclase. To activity, of

we

fatty

late

acyl

have

effects developped

chains

cyclase

activities.

linoleate

and

glucagon

-

MATERIAL

AND

of

modifications

a technique

incorporation The

arachidonate

stimulated

such

for

into present

the

the study

incorporations adenylate

upon

simultaneous

compares basal,

and the

cyclase measurement

phospholipids

on

cyclase

adenylate

effect

fluoride

of

the

of

adeny-

oleate,

- and

activities.

METHODS

- Linoleic acid, oleyl-CoA, coenzyme A, ATP, dithiothreitol, EDTA were obtained from Sigma. Cyclic AMP, creatine phosphate kinase were from Boehringer-Mannheim. [l-l'+C] oleyl-CoA (50 mCi/mM), linoleic acid (51 mCi/mM), acid (55.5 mCi/mM), cyclic [aH] AMP (39.8 Ci/mM) and [c(-~~P] were purchased from New England Nuclear.

and

albumin creatine

arachidonic ATP (20

and

Ci/mM)

- [1-14C] linoleyl-CoA and [l-14C]-arachidonyl-CoA thioesters were prepared using the mixed anhydride method described by Wieland and Rueff (16) and purified according to Seuberg (17). The specific activities used were 10 pCi/umole. Their ultraviolet spectrum were typical of acyl-CoA derivatives and exhibited a maximal absorbance at 257 nm and a A2a2/A2s7 ratio ranging from 0.53 to 0.62 (theoretical ratio 0.55). The yields of synthesis were between 40 and 60 % when based on fatty acid incorporation in the thioester. - Plasma membranes were prepared from male Wistar rats weighing 200 g according to the procedure devised by Neville up to step 12 (18). The fraction collected above the sucrose gradient layer of density 1.19 was resuspended in 1 mM NaHCOs and centrifuged at 25.000 x g for 15 min. The membrane fraction (0.6-0.9 mg protein/g liver) was stored in liquid nitrogen. Protein determinationswere done according to Lowry et al (19). - Transacylation and adenylate cyclase simultaneous measurements. Transac lase activity was measured by the incorporation of['4C] acyl chains from [ Y '+C] acyl-CoA thioesters into the plasma membrane phosphatidylcholine and phosphatidylethanolamine. Adenylate cyclase activity was measured by the conversion of [cx-~*P] ATP into cyclic I 32F] AMP. The standard reaction mixture (0.1 ml) contained : 50 mM Tris-HCl pH 7.5; 5 mM MgCl,; 1 mM ATP, dithiothreitol, 10 viM cyclic AMP ; 1 mM EDTA ; U.5 mM ATP and [c~-j'P] acyl CoA thioester at varying concentration, U.5 % (w/v) about 1 UCi ; [l'+Cl albumin and o.4 mg/ml membrane protein. In addition, creatine kinase (50 units) and phosphocreatine (5 mM) were added as an ATP regenerating system. The reaction was initiated by the addition of membranes and carried out at 30°C for 10 min. Reaction was stopped by cooling in ice and by diluting with 1 ml pH 4-5. Samples were then centrifuged at 1 600 x g for 15 min. CAMP HzS04, was purified from the supernatant according to Salomon et al. (20) using a sequel tial chromatography on Dowex and alumina columns, cyclic [31i] AMP being added at the begining of the purification step to monitore cyclic 13*P] AMP recovery.

98

Vol.

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BIOCHEMICAL

1, 1980

AND

BIOPHYSICAL

RESEARCH

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The membrane pellet was extracted with 1 ml chloroform-methanol (l/l ; v/v). Organic phase was then dried under Nz and lipids spotted on silicagel G plates. Chromatography was carried out in the following system ; chloroformmethanol-acetic acid-water (25/15/4/2)(21). Spots visualized by exposure to iodine vapor or by autoradiography were scrapped and 0.5 ml of methanol was added to each counting vial before adding 2.5 ml of scintillation fluid.

RESULTS In

AND

DISCUSSION

our

membrane

preparations,

genous

phosphatidylcholine

higher

when in

conditions with

linoleic

The levels

be

membrane were

donor

in

linoleyl

remained

low

when CoA

The

fluoride

Incorporation

of

increased

tory to

and

effect its

appears

different

phospholipids

the

variations

of

the

transacylated

related the

adenylate fatty

was

oleyl

at

the

about

linear

best

CoA

was

rates

25

up to also

measured

at

UM least

linear

but

using

either

a good

not

linoleate

adenylate

cyclase

3 nmoles/

mg

of by

the

the

%.

produce

any

variance

For

CoA

the

of

highest

be

this

itself

noted

inhibi-

and that

not

the

same

inhibition.

there

incorporation is

and to

alter protein

Thus

arachidonate

activities

not

incorporation

arachidonyl can

increase activities.

did

decreased.

It

99

50

the

the

the

3 nmoles/mg

arachidonate

activities

that

at

protein,

incorporation

on

between

stimulated

and

cyclase

correlation

activity

to

depended

and

demonstrated

acids

was

phospholipids

phospholipids. did

of

CoA

was

phosphatidylethanolamine.

simultaneously

tested,

cyclase

oleyl-CoA

clearly

to the

CoA

be

effects

in

in

arachidonyl CoA

membrane

cyclase

into in

the

up

adenylate to

-

while

arachidonyl

incorporation

concentration The

the

There the

adenylate

of

than

activities

and

occured

measured

glucagon

activity

CoA

acylation

CoA

transacylation

into

and

The

thioesters.

activity

arachidonate,

linoleate

as

from

the

2).

I).

incubation

linoleyl

arachidonyl

incorporation

CoA

(fig.

-

cyclase

concentrations

to

linoleate

adenylate

linoleyl

well

transacylation

linoleyl

tested of

plateaued

The

fold

(Table

same

cyclase

endo-

ten

phosphatidylethanolamine

CoA,

linoleate

cyclase

thioester

basal,

the

COA the

of

adenylate

concentration, as

compared or

adenylate

incorporation in

oleyl low

presence in

about

and

under

the

incorporation. and

At

acid

lysophospholipid-acyltransferase while CoA.

arachidonyl

acyl

the

CoA

$I

acyl

phosphatidylcholine

of

arachidonyl

the

increases

the

increasing 1).

into was

linoleic

in

phosphatidylcholine

(fig.

incorporation

measured

higher

these to

measured

Saturation

100

Thus,

with

activities

fold

related

concentrations acyl

ten

acid. to

than

cyclase

also

linoleate

phosohatidylethanolamine

linoleyl-CoA

adenylate

were

appeared

and

using

increases

the

what

no 1)

the had

into

relation

between

unsaturation been

described

degree for

II

I

Exp.

mM linoleyl

CoA

6.7OfO.32

4.7OkO.26

322i24 (x2.5)

4000243 (x2.5)

1584i44

127213

0.1

(x1.1)

none

3089klY (x1.2)

0.35kO.04

228i2

0.74icJ.04

0.1 mM linoleic t 0.1 mM CoA

acid

2795+11

cyclase min/

18425

Adenylate (pmoles/lO

none

min/mg protein incorporated into Phosphatidylethanolamine

tNaF

nmoles/lG Linoleate Phosphatidylcholine

either of two upon

2084+15 (x2.1)

952+4

(x1.25)

1640254

1308+5

tglucagon

activity mg proteinl_

(40 ug) were incubated in the standard reaction mixture with or without CoA or G.1 mM [1-14C1 linoleic acid plus 0.1 mM CoA. Values are the mean between brackets represent the fold increase in adenylate cyclase activity CoA additions. [NaF] was 10 mM and [glucagonl was 1 PM.

basal

Addition

TABLE I. Membrane protein 6.1 mM [1-14C] linoleyl determinations. Numbers linoleic acid or linoleyl

Vol.

95,

No.

BIOCHEMICAL

1, 1980

E 52 .

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

PE

25

50 Acyl

100 CoA (FM)

50

25

100

FIGURE 1. Fatty acyls incorporation into plasma membrane phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from various acyl-CoA thioesters. Transacylase activities were determined in the standard reaction mixture. All the values are the mean of two determinations. N oleyl CoA, l elinoleyl CoA, o--O arachidonyl CoA.

0.4 (25)

0.7 150)

OLEATE

1.4 (6.2) LINOLEATE

1.2 (100)

incorporated

-----a 0

5.3 (25) incorporated

6

-50 1 1.75 (6.2) ARACHIDONATE

(1235) mcorporated

(254(z) (100)

FIGURE 2. Relation between the incorporation of the various acyl groups into the phospholipids and the adenylate cyclase activity. The acyl group incorporations are those of PC + PE reported in figure 1 and are expressed in nmoles/ 10 min/mg protein. The numbers in parentheses refer to the UM concentration values for which these incorporations were obtained. Transacylation and adenylate cyclase measurements were made simultaneously as described under material and methods. The percent change in adenylate cyclase activity was plotted as a function of the amount of acyl incorporated into the membrane phospholipids. O---O basal, N NaF-stimulated and Aglucagon-stimulated adenylate cyclase activity.

101

vol.

95.

No.

BIOCHEMICAL

1. 1980

the

PGEl-stimulated

the

lysophospholipid

being

known

activities

adenylate level

to

inhibit

fluidity.

occur

at

used

and/or

brane.

two

In

the

may

species

chemical

properties.

cally

the

correlated

was

to

hypothesis

the

carry

the

synthesis

the

the

decrease

in

adenylate

into

the as

of areas

cyclase plasma

a whole

reacylation the of

acyl the

thioester mem-

in

the

membranes

molecular

might

CoA

phospholipid

plasma

the

plasma

Z-linoleyl

stimulation

memof

process

of

cyclase

phospholipid

COMMUNICATIONS

lysophosphatidylcholine Thus

dependent

changes

adenylate

the

specific

formation

of

2)

a modification

be

some

local the

11).

of

may

(8),

incorporated

by

specificity : it

RESEARCH

fraction, (5,

explained

levels

Anyhow to

LM cells

membrane

restricted

could

in

linoleate

However

be

BIOPHYSICAL

cyclase

be

latter

molecular

the

cannot

different it

in

when

phospholipids

membrane

cyclase

adenylate

enhancement

brane

AND

was species

physic0 specificontainin<

linoleate. ACKNOWLEDGEMENTS This

work

was

supported

by

D.R.E.T.

78 34

020

00 480

75

01

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

POHL, S.L., KRANS, H.M.J., KOZIREFF, V., BIRNBAUMER, L. and RODBELL, M. (1971) J. Biol. Chem. 246, 4447-4454. RUBALCAVA, B. and RODBELL, M. (1973) J. Biol. Chem. 248, 3831-3837. LAD, P.M., PRESTON, M.S., WELTON, A.F., NIELSEN, T.B. and RODBELL, M. (1979) Biochim. Biophys. Acta 551, 368-381. RETHY, A., TOMASI, V., TREVISANI, A. and BARNABEI, G. (1972) Biochim. Biophys. Acta 290, 58-69. SHIER, W.T., BALDWIN, J.H., NILSEN-HAMILTON, M., HAMILTON, R-T. and TMANASSI, N.M. (1976) Proc. Natl. Acad. Sci. USA 73, 1586-1590. HOUSLAY, M.D., HESKETH, T.R., SMITH, G.A. and METCALFE, J.C. (1976) Biochim. Biophys. Acta 436, 495-504. STORM, D.R. and GLASER, M. (1976) Proc. ENGELHARD, V.H., ESKO, J.D., Natl. Acad. Sci. USA 73, 4482-4487. ENGELHARD, V.H., GLASER, M. and STORM, D.R. (1978) Biochemistry 17, 3151-32OD. HIRATA, F., STRITTMATTER, W.J. and AXELROD, J. (1979) Proc. Natl. Acad. Sci. USA 76, 368-372. ZWILLER, J., CLESIELSKI-TRESKA, J. and MANDEL, P. (1976) FEBS Lett. 69, 286-250. HOUSLAY, M.D. and PALMER, R.W. (1979) Biochem. J. 178, 217-221. VICTORIA, E.J., VAN GOLDE, L.M.G., HOSTETLER, R.Y., SHERPhOF, G.L., VAN DEENEN, L.L.M. (1971) Biochim. Biophys. Acta 239, 443-457. NEWKIRK, J.D., WAITE, M, (1973) Biochim. Biophys. Acta 298, 562-576. COLARD-TORQUEBIAU, O., WOLF, C. and BEREZIAT, G. (1976) Biochimie 58, 587-592. STAHL, W.L. and TRAMS, E.G. (1968) Biochim. Biophys. Acta 163, 459-471. WIELAND, T. and RUEFF, L. (1953) Angew. Chem. 65, 186-187. SEUBERG, W. (1959) Biochem. Prep. 7, 80-83. NEVILLE, D.M. (1968) Biochim. Biophys. Acta 154, 540-551. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. (1951) J. Biol. Chem. 193, 265-275. SALOMON, Y., LONDOS, C. and RODBELL, M. (1974 ) Ann. Biochem. 58, 54 l-548. GRAY, G.M. (1967) Biochim. Biophys. Acta 144, 511-515.

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