Divergent effects of propranolol on neutrophil superoxide release: Involvement of phosphatidic acid and diacylglycerol as second messengers

Vol.

175.

March

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

15, 1991

DIVERGENT

423-429

EFFECTS OF PROPRANOLOL ON NEUTROPHIL

SUPEROXIDE RELEASE:

INVOLVEMENT

OF PHOSPHATIDIC

ACID

AND DIACYLGLYCEROL AS SECOND MESSENGERS Denis

English

Departments Indiana

and Gregory of Medicine

University

November

30,

and Pathology

School

Indianapolis, Received

S. Taylor

of Medicine

IN

46202

1990

SUMMARY Relatively high levels of propranolol(170 PM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils the drug had only a slight inhibitory influence on treated with FMLP alone, diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with propranolol induced a enhancement of FMLP-induced O,- generation. However, similar dose-dependent inhibition of O,- generation in neutrophils stimulated These results are with either FMLP + cytochalasin B or with 20.0 mM NaF. consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil Oz-release. @1990 Academic PESS, rnc INTRODUCTION presence been

The

and absence

adequately almost

responses

of

activation

period

again

(or

(1).

added

While

Neutrophils B markedly acid

in

human

of lo-20

with

similar

formyl changes

diglyceride

mM) effects

neutrophils

(5).

the

oxidative

have been in

in phospholipid

F--treated

an extended when 0,.

in

begins

remains

levels,

in FMLP-stimulated accumulation

or but

in

obscure. absence

of

cytochalasin

neutrophils

(3).

of phosphatidic phosphatidic 0006-291X/91

423

an

to alterations

presence

neutrophils,

after

response

basis

the

FMLP

minutes,

generating

attributed

biochemical

peptides

for

have not

Interestingly,

FMLP alone,

a pronounced In

1).

the

by

B commence

ceased

their

that 2-3

and continue

figure

FMLP in

induced

approximately

cytochalasin

generation

Fe (20.0

reasons

have

(2),

to

release

that

effects

to

after

also

neutrophils

for O,-

seconds

see

interactions exposed

enhances

FMLP (4),

ceases

some of these

B display

Like

to

while

with

(1,

stimulation

receptor-cytoskeleton cytochalasin

phase)

minutes)

B is to prior

and

exposed

markedly,

example,

pretreated lag

neutrophils

B vary

For

immediately

(lo-20

cytochalasin

of

of cytochalasin

neutrophils

interval

response

responses

explained.

commences

time

metabolic

acid $1.50

Copyright 0 1991 by Academic Press, IM. All righis of reproduction in any form reserved.

Vol.

175,

No.

generation

precedes

initiation

is

effect

expanded

in

this inhibits

suggesting

that

present

study,

the and

to in in

cytochalasin

the

leading

stimulated

acid

are and

the

to activation with

FMLP in

as

of phosphatidic data,

which

propranolol

are

almost

acid

generated

in neutrophils

a

indicate optimal

treated that

both of

biochemical

superoxide

presence

of

responses

fundamental

of the the

as

of the

consequences

The results for

acid

The purpose

functional

required identify

B

phosphatidic

D (7).

of PA and diacylglycerol

stimuli

pathways

by

of phosphatidic

of phospholipase

phosphatidic

diglyceride

phosphohydrolase

that

from

B and NaF.

that

in F--treatedneutrophils,

derived

document

in

of cytochalasin

recovery

of diacylglycerol

to

inhibits

acid the

involved

demonstrated

presence

demonstrate

is

was

have

be

Our preliminary

dephosphorylation

metabolic

neutrophils

in

cells.

FMLP + cytochalasin

diacylglycerol difference

increase similarly

equilibrium

the

COMMUNICATIONS

may

markedly

FMLP in

of phosphatidic

activation

therefore,

FMLP alone,

enzyme

with

of stimulated

catalysed

of F--mediated

neutrophils

reports

diacylglycerol

a result

in

Recent

the accumulation

phosphohydrolase

alterations

(5).

concentrations,

communication,

completely

with

therefore

treated

extracts

RESEARCH

and

by a corresponding

in organic

BIOPHYSICAL

release

is due to inhibition

accompanied

acid

high

in neutrophils

This

AND

activation

at relatively

generation it

superoxide

of oxidative

propranolol,

(6).

BIOCHEMICAL

2, 1991

and

generating absence

of

B.

MATERIALS AND METHODS NaF, FMLP, cytochalasin B, cytochrome C and other reagents and buffers were from Sigma Chemical Co., St. Louis, MO. Radioactive compounds were purchased from New England Nuclear, unless otherwise noted. Organic solvents used for lipid extraction and thin layer chromatography were from Fisher Scientific, Pittsburgh, PA. Neutrophils were isolated from human blood by enhanced sedimentation, ficoll-Hypaque density gradient centrifugation and erythrocyte Lysis as previously described (1). Cells were resuspended in HEPES (10.0 mM) buffered saline (pH 7.4) containing 1.0 mM CaCl, unless noted otherwise. Cells were suspended at a concentration of 2-3 x 107/ml for isotopic labeling. Labeling was accomplished by incubation for 60 min at 37'C with 32P-orthophosphate (1.0 mCi/ml) or 3H-glycerol (100 &i/ml). After incubation, cells were washed twice in HEPES buffered saline and resuspended at a concentration of 2-3 x LO' cells/ml. Neutrophil superoxide release was monitored continuously in Perkin-Elmer thermostatted (37Y) spectrophotometer in the presence of cytochrome C as previously described (1). Phospholipids in labeled cells were analysed by thin layer chromatography of organic extracts using a solvent system consisting of chloroform:methanoL:20% aqueous methylamine (60:35:10), as previously described (4,5). Diglycerides were determined by the mass assay method of Preiss et al (8). Differentiation of 1,2 diacylglycerol from 1-O-alkyl,2-acylglycerol was accomplished by preincubation of extracts with Rhizopus phospholipase A,, as described by Tyagi et al (9). Over 90% of the diglyceride recovered in cells stimulated with either F‘ or with FMLP was determined to consist of 1,2 diacylglyceride. In some experiments, diglycerides were measuredby radiometric quantitation of labeled diglycerides separated by thin layer chromatography of organic extracts of cells prelabeled with 3H-glycerol. The solvent system consisted ofhexane:ether:gO% formic acid (90:60:6). Extracts were "spiked" with Sigma) to enhance visualization 100 pg of unlabeled mixed diglycerides (diolein, Chromatographs were exposed to iodine vapor and the spots after chromatography. 424

Vol.

175,

No.

2, 1991

corresponding containing radioactivity RESULTS

10

release

pretreatment

by

with

pretreated

with

M FMLP.

Figure

triggered

release

triggered

BIOPHYSICAL

1 contrasts

neutrophils

5pg/ml

B.

For

cytochalasin

by

the

stimulated

cytochalasin

In the absence

release

AND

RESEARCH

to 1,2 and 1,3 diacylglycerol were mls of aqueous liquid scintillation by scintillation counting.

AND DISCUSSION

superoxide

lo-'

BIOCHEMICAL

FMLP.

In

by exposure

latter

prior

with

to

cells

were

stimulation

with

enhanced

strongly

B-pretreated

on

FMLP after

markedly

propranolol

of cytochalasin

propranolol

determinations,

B, propranolol

contrast,

of

FMLP and

2 minutes

of cytochalasin

dropped into vials and assayed for

influence

with

the

B for

excised, fluid

COMMUNICATIONS

inhibited

cells

0,. 0,.

to FMLP.

The

22c

210

E. FMLP Alone

200

190 160

170 ii

160

0i?

160

% :

140

6

130

E E

120

k

110

s

100

3

so

/ 0

1

2

3 TIME

D. Cytochalasin

5 g

80

‘N 0

70

4

5

1 6

5

6

(min)

B FMLP

60

60

40

30

20

10

0

Cylochalarin (Total

I 20

u 40

I 60

I 60

I 100

PROPRANOLOL

J.

B+FMLP Response)

I

I

I

I

I

I

I

I

120

140

160

180

200

220

240

260

CONCENTRATION

((IM)

, 0

I 1

2

3

TIME

4

(mid

Fieure 1. Divergent effects of propranolol on FMLP-stimulated 0; release in untreated (A,B) and cytochalasin B pretreated (C,D) neutrophils. Panels B and D are traces of individual spectrophotometric recordings; the data of panels A and C were derived from 3 determinations. In panels C and D, cells were pretreated with 5 pg/ml cytochalasin B two minutes prior to stimulation with 1O-7 M FMLP. 42.5

Vol.

BIOCHEMICAL

175, No. 2, 1991

similarity

of

the

was striking;

dose-dependencies

maximal cells

was

inhibition

of O,- release

observed

of the dose-response

curves

is

with

propranolol

increased

O,- release.

both

At higher

triggered

had no effect

is

the

the

a known

rate

inhibitor

of

(6,10,11),

acid

activation

of the neutrophil

NADPH oxidase

a cell

free

enhancement cytochalasin

B correlated

acid

effected

levels

However,

maximally

at

generation that

the majority derived

diacylglycerol (Table

with

susceptibility but

this

effect

of

untreated

neutrophils In

is

contrast,

generation

of

inhibition

of phosphatidic

acid

increases

to propranolol

the divergent and

2).

suggests

B and this

generation

the basis

by

result

B pretreatment

diglyceride

with

FMLP-induced

acid.

in the recovery

of

(figure

This

cytochalasin

degree

of phosphatidic

inhibited I).

in and

pretreated

FMLP-induced

cytochalasin

may explain release

with

increase

by which

FMLP-dependent

superoxide

the

inhibited

pretreated

not

propranolol

phosphatidic

either

messenger The

in FMLP-stimulated

of

a corresponding

of

on

generated

markedly

The mechanism

I).

clear,

pm)

in neutrophils

was associated

10% (Table

release

but

(6,7,12,13)

enhancement

than

by less

0,.

phosphohydrolase

cells were

of

propranolol

dephosphorylation

(170

of

doses,

response.

acid

that

extent

of

(14-16).

levels,

of diacylglycerol from

propranolol

drug

with

of diacylglycerol

not

in cells

were

of FMLP-induced

as a second

system

concentrations

stimulating

oxidative

in intact

The

that

At lower

duration

phosphatidic

strongly

the divergent

of O,- release,

implicated

varying

similarity

cells

extent

the the

in

effect.

extent

both

maximal

on a common process. O,- release

activation

O,- release

by

that

of

has been

NADPH oxidase

of FMLP-induced

the hypothesis

increased

and phosphatidic

in

The strong

the total

initial

caused

cells.

and the total

drug

by previously

that

an unusual

rate

of propranolol

O,- release

influence

B displayed

increasing

effects

propranolol

with its

the initial

or inhibited

Propranolol

of

of FMLP-triggered

doses,

by FMLP, thereby

divergent

B treated

from

cytochalasin

RESEARCH COMMUNICATIONS

FMLP triggered

levels

consistent

resulted

of enhancement

not pretreated

of

with

these

in cytochalasin

of propranolol

characteristics

of

enhancement

untreated

effects

AND BIOPHYSICAL

influence

cytochalasin

the is

not

of

the

B-pretreated

neutrophils. At

concentrations

generation that

of

of 1,2 cytochalasin

to be highly it

demonstrated on

O,-

inhibition neutrophils

from

in figure

release was

neutrophils

3, propranolol

by similar

to with

the

stimulated

with

exerted

acid

a strong

dose-dependency

FMLP after

acid

phosphatidic

neutrophils. pretreatment 426

induces

response, FMLP,

(Table

I),

was found with

suggesting

of cytochalasin

As

inhibition

dose-dependency

inhibition with

the like

phosphohydrolase.

dose-dependent

The of

This

and was associated

of phosphatidic of

F-

release,

(7).

by propranolol

in recovery inhibition

F- stimulated

stimulated

superoxide

in human neutrophils

to inhibition

increase

results

stimulate

B pretreated

susceptible

a corresponding that

which

diacylglycerol

0,'

of

this

release B (Figure

of

Vol.

175,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

200

I

I

I

200

I

1000

INCREASE

2000

IN PA - ASSOCIATED (CPM)

RADIOACTIVITY

Figure 2. Correlation of enhancement of phosphatidic acid levels and 0,. release in FMLP-treated neutrophils. Cells were prelabeled with 3H-glycerol and stimulated with 10.' M FMLP. Identical portions of cells were used to assay 0; generation during FMLP stimulation and diglyceride levels after stimulation. Thus, after 0; release ended, the prelabeled cells were extracted with chloroform/methanol/HCl, spikedwith unlabeled diglyceride and the organic phases separated in a solvent system consisting of hexane:ether:gO% formic acid (90:60:6). Bands corresponding to the migration position of 1,2 diacylglycerol were excised, dropped into vials containing scintillation fluid and assayed for radioactivity by liquid scintillation counting.

with enhancement of O,- release in neutrophils l), (figure 1) and with inhibition of diglyceride generation

treated

with

shown).

either In

F-or

with

FMLP + cytochalasin propranolol

summary,

neutrophil

oxidative

metabolism,

strongly

inhibits

in cells

stimulated

mM NaF.

On the other

hand,

and this

enhancement

correlates

acid.

responses with

However,

NADPH

oxidase

activation

in

Diglyceride

generation

propranolol, that

both

activation

as it

cells

while

diminishes

with

cannot

its

and

by

with these

in response phosphatidic

NADPH oxidase. 427

effects

on

stimulus

used.

enhanced be the

also

Fe or with agonists

is are

to

FMLP

20.0 alone

of phosphatidic

messenger

involved

in

markedly

enhanced

by

FMLP + cytochalasin

B.

strongly

by

to FMLP alone acid

generation B or with

responses

generation

of

Propranolol

diacylglycerol

second is

treated

initiation

cytochalasin

enhances

generation

either

generated

with

markedly

alone

induced

neutrophil

pretreatment

closely

since

diacylglycerol

of the

markedly

propranolol

stimulated that

on the

FMLP alone

in neutrophils

not

divergent

depending

FMLP after

phosphatidic

propranolol

B (data

exerts

with

is not. required

inhibited We conclude for

optimal

Vol.

175, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect Levels in

170 pM Propranolol

Stimulus

of Propranolol Neutrophils

I

on Diacylglycerol Treated with F-,

Diacylglycerol nmol/l.2 x 10'

FMLP

and Phosphatidic Acid and FMLP + Cytochalasin

'1. Inhibition of stimulated increase

CSllS

Phosphatidic acid

105 *

2.41

+

236 225

k +

9.18 9.3

+

587 241

f +

45.9 19.0

71.8

+

761 173

f *

14.23 9.1

09.7

125.3 144.5

NOTIf?

FMLP

FMLP + CB

Fluoride

B

% Enhancement of stimulated increase

100

a.4

116.4

f 4.2

122.1

+ 3.1

115.6

f

131.1

f 3.9

99.4

+ 4.5 + 6.2

75.8

34.9

7.1

Diacylglycerol mass levels were determined as described by Preiss et al. using the (8) modifications of Tyagi et al. (9). Phosphatidic acid levels were determined by quantitation of radioactivity recovered with phosphatidic acid in thin layer chromatographs of organic extracts of 3H-glycerol labeled neutrophils. Results are expressed in reference to the radioactivity recovered with phosphatidic acid in untreated cells, which was assigned a value of 100. Similar results were observed when diglycerides were quantitated radiometrically (with 3H-glycerol labeled cells)and when phosphatidic acid was monitored with cells prelabeled with 32P-orthophosphate. As indicated, cells were either not stimulated or exposed to 10W7M FMLP (FMLP), 5 pg/ml cytochalasin B followed by 1O-7 M FMLP (FMLP + CB) or 20 mM sodium fluoride for 10 minutes at 37'C. Lipids were then extracted and analysed as indicated in the methods. The % inhibition or enhancement of stimulated increases refers to the portion of the increase that was susceptible to inhibition or enhancement by 170 PM propranolol.

100 90 80

0 20

40

60

PROPRANOLOL

80

100

120

CONCENTRATION

140

160

180

@M)

Fiaure 3. Inhibition of F-- stimulated O,- release by propranolol. Results are derived from 3 determinations, showing the mean and standard deviation for each concentration of propranolol. 0; release was measured as the initial rate, approximately 8.5 minutes after addition of 20.0 mM NaF. The control response in this experiment was 3.2 nmoles Oz./l.2 x 10' cells/min.

428

Vol.

175,

No.

ACKNOWLEDGMENTS a grant-in-aid by a grant for

BIOCHEMICAL

2, 1991

This from

from

preparing

the

investigation Indiana

the Phi Beta the

Psi

AND

BIOPHYSICAL

was supported Affiliate

RESEARCH

by NIH grant

of the American

Sorority.

COMMUNICATIONS

The authors

Heart thank

{/ AI-25656, Association

Stephanie

by and

McGillem

manuscript.

REFERENCES 1. 2. 3. 4.

English, D., Roloff, J., and Lukens, J.N. (1981) J. Immunol. 126, 1656-1661. Jesaitis, A.J., Tolley, J.O., Painter, R.G., Sklar, L.A., and Cochrane, C.G. (1985) J. Cell. Biochem. 27: 241-253. Truett, A.P. III, Verghese, M.W., Dillon, S.B., and Snyderman, R. (1988) Proc. Natl. Acnd. Sci. (USA) 85, 1549-1553. Volpi, M., Yassin, R., Naccache, P.H., and Sha'afi, R.I. (1983) Biochem. Biophys. Res. Comm.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

11,

957.964.

English, D., Debono, D.J.. and Gabig, T.G. (1987) J. Clin. Invest. 80:, 145.153. Billah, M.M., Eckel, S., Mullman, T.J., Egan, R.W., and Siegel, M.I. (1989) J. Biol. Chetn. 264: 17069-17071. English, D., and Taylor G. (1990) Clin. Res. 38, '245A (abstract). Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E., and Bell, R.M. (1986) .I. Biol. Chem. 261, 8597-8601. Tyagi, S.R., Burnham, D.N., Lambeth J.D. (1989) J. Biol. Chem. 264, 12977-12981. Pappu, A.S., and Hawser, G. (1983) Neurochem. Res. 8, 1565-1575. Koul, O., and Hauser, G. (1987) Arch. Biochem. Biophys. 253, 453.461. Rossi, F., Grezeskowiak, M., Della Bianca, V., Calzetti, F., and Gandini, G. (1990) Biochem Biophys. Res. Comm. 168, 320-327. Boxer, R.W., Thompson, N.T., Randall, R.W., and Gerland, L.G. (1989) Biochem. J. 264: 617. 620. Bellavite, P., Corso, F., Dusi, S., Grzeskowiak, M., Della-Bianca, V., and Rossi, F. (1988) J. Biol. Chem. 263: 8210-8214. Ohtsuka, T., Ozawa, M., Okamura, N., and Ishibashi. S. (1989) .I. Biochem. 106, 259.263. Peveri, P., and Curnutte, J.T. (1990) Blood 76 (suppl). 109a (abstract).

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