BIOCHEMICAL

Vol. 90, No. 4, 1979 October 29, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1364-1370

SECRETAGOGUES FOR LYSOSOMAL ENZYME RELEASE AS STIMULANTS OF ARACHIDONYL PHOSPHATIDYLINOSITOL

TURNOVER

IN RABBIT NEUTROPHILS R.P.

Rubin,

L.E. Sink, M.P. Schrey, C.S. Liao, and R.J. Freer

A.R.

Day,

Department of Pharmacology Medical College of Virginia Richmond, Virginia 23298 USA Received

September

17,

1979 SUMMARY

[14C]arachidonic acid incorporation into phosphatidylinositol from rabbit neutrophils pretreated with cytochalasin B was increased within 2 synthetic peptide formyl-methionyl-leucyl-phenylalanine and the Ca !!Jni~o$~re A23187. A high concentration of the pe tide elicited only a small increase in phosphatidylinositol turnover when [ a4 Cjpalmitic acid was employed as pr cursor. These data coincide with our earlier studies which identified a increase in arachidonyl phosphatidylinositol turnover during Ca f+ -dependent adrenocorticotropin and A23187-induced activation of steroid production and in arachirelease in adrenocortical cells. It is concluded th t an increase phospholipase donyl phosphatidylinositol turnover mediated by a Ca 8+ -dependent A2 may be a general mechanism for altering membrane function during secretory activity. INTRODUCTION There

is

phospholipase

(l),

involved

with

of arachidonic and one or all

secretory

process.

requires

Ca2+ for

enzyme

(2)

secretion event

recognition

A2 in such cellular

principally release

an increasing

since

Abbreviations:

acid,

functions

The fact

that

Ca2+ plays (3).

might

and critical

The elucidation

can be correlated

with

secretory

role

activity

1364

the

synthesis the

A2 absolutely of this in stimulusbiochemical

is of prime

ACTH = adrenocorticotropin; PI = phosphatidylinositol; F-Met-Leu-Phe = formyl-methionyl-leucyl-phenylalanine; Met-Leu-Phe = methionyl-leucyl-phenylalanine.

@ 1979 by Academic Press. Inc. in any form reserved.

of reproduction

enzyme is

activating

importance

of a Ca2+-dependent

0006-291X/79/201364-07$01.00/0 Copyright All rights

for

of

phospholipids,

phospholipase

the potential

a ubiquitous

This

of prostaglandin

be crucial

non-lysosomal

heightens

importance

of membrane

and the enhancement events

potential

as secretion.

transformation

of these

activity

coupling

which

the

of the

importance

Vol. 90, No. 4, 1979

before

the role

laboratory

BIOCHEMICAL

of Ca*+ can be defined.

has demonstrated

dependent

activation

turnover reacylation

sequence

activation

of steroid

Neutrophils since

on the

cell

lysosomal peptide vation which

in adrenocortical

they

A2 (4),

phosphatidylinositol was temporally release

can also

surface

respond

of arachidonic resembles

work

cells

that

which

brings

from

this

ACTH causes

This

and quantitatively

as a model

to chemotactic

(7,8).

for

about

a Ca*+-

an increased

deacylation-

linked

turnover

investigating

peptides A23187

The present

study

and the Ca*+ ionophore acid

regard,

(5,6).

and to the Ca*+ ionophore

F-Met-Leu-Phe

RESEARCH COMMUNICATIONS

to ACTH-induced

(6).

serve

enzyme release

closely

In this

of phospholipase

of arachidonyl

process

AND BIOPHYSICAL

in the

PI fraction

secretory

interacting

with

with

an enhancement

reports

A23187

the deacylation-reacylation

the

that

produce

rabbit

sequence

of

the synthetic

an early

from

receptors

actileucocytes

in adrenocortical

cells.

MATERIALS

AND METHODS

Neutrophils were obtained from albino rabbits injected intraperitoneally with 400 ml sterile isotonic saline containing 0.1% glycogen and the peritoneal exudate was collected in heparinized flasks 12 hr later [for details see (9)]. The cells were washed with buffered ammonium chloride to hemolyze erythrocytes and then resuspended in a Modified Earle's Balanced Salt solution containing 0.5 mM Ca*+, 0.2 mM Mg* +, 20 mM Hepes (pH 7.4), and 0.025% bovine serum albumin. This medium was used throughout the remainder of the experiment. The neutrophils were incubated with c tochalasin B (5 l.~ /ml) for 15 min and then added to incubation tubes (4 x 10 % cells/incubation (0.2 uCi) [14C]7 containing arachidonic acid or [14C]palmitic acid in the presence or absence of stimulating agent. The final reaction volume was 500 ~1. Incubations were carried out at 37°C for various time intervals. The cellular incorporation of radiolabel was terminated by adding 3 ml of chloroform/methanol (1:2) and the lipid was extracted overnight. The lipid was subjected to thin layer chromatography using a two-dimensional system as described elsewhere (6). The areas corresponding to each phospholipid standard were scraped into vials and counted for 10 min by liquid scintillation spectrometry. All values were subtracted from background counts which averaged 34(?1) cpm. Statistical analysis was carried out by paired Student t test. F-Met-Leu-Phe and its unformylated derivative were pr ared as described previously (8). [14C] arachidonic acid (56 mCi/mmol) and [ Clpalmitic acid (59 mCi/mmol) were obtained from Amersham Radiochemical Center. The calcium ionophore A23187 was generously provided by Eli Lilly Company. Phosphatidylinositol was obtained from Supelco, Inc.; all other phospholipid standards and cytochalasin B were obtained from Sigma Chemical Company.

74

1365

Vol.

90,

No.

4, 1979

BIOCHEMICAL

754)

AND

I 10-f’

. 1.

Comparison

RESEARCH

3x104

COMMUNICATIONS

lo-‘0

1-

-

Log

Fi

BIOPHYSICAL

[ F-Mel-Leu-Phe

Molar

the fffect of F-Met-Leu-Phe on the incorporation and [ 'klpalmitic acid into phosphatidylinositol rabbit neutrophils. Neutrophils pretreated with cytochalasin were incubated for 2 min with either radiolabeled arachidonic or palmitic acid in the presence or absence of varying concentrations of F-Met-Leu-Phe and processed as described in Methods. All values are means (tS.E.1 obtained from duplicate determinations from 3-5 different experiments. C arachidonic d-lderived from

of

of

acid

RESULTS When neutrophils plus

F-Met-Leu-Phe,

in the in

were a dose

incubated related

PI fraction

was observed

[14C]arachidonate

incorporation

F-Met-Leu-Phe lo-lo further

increases

preincubated

in label

[14C]arachidonic

acid

nificant

in the

into

the

although

There

into

PI after

~~0.05);

maximal

with

phosphatidylserine

(data

of radiolabel

was a significant

increase

effects

not lo-lo

were

shown).

fraction

this

1366

Whencells

M

with no were

enhanced

(n=3).

No sig-

phosphatidylserine, range

arachidonic

was increased

M produced

M F-Met-Leu-Phe

of phosphatidylcholine,

M F-Met-Leu-Phe,

to 10-l-l

observed

low9

of control

over

acid

incorporation

a 2 min exposure

by 133(+10)X

were manifest lo-10

of [14C]arachidonic

F-Met-Leu-Phe

of cytochalasin,

incorporation

and phosphatidylethanolamine trations,

1).

incorporation

labeling

presence in the

(207% of control).

in the absence

changes

increase (Fig.

(114% of control,

M F-Met-Leu-Phe

in the

of peptide acid

by 37(+21)%

concen-

incorporation (p=O.O5)(n=3).

BIOCHEMICAL

Vol. 90, No. 4, 1979

AND BIOPHYSICAL

Table

RESEARCH COMMUNICATIONS

1

Effects of F-Met-Leu-Phe, Met-Leu-Phe and A23187 on the labeling of phosphatidylinositol with [14C]arachidonic Radiolabeled

(lOWIOM)

Met-Leu-Phe

10 min -

30 min

207 f 21*

163 2 22*

128 + 16

(lO-l"M)

88?

A23187 (5uM)

PI (% of Control)

2 min

Agent F-Met-Leu-Phe

acid

1

106f

373 + 60*

7

91 f

335 2 58*

8

301+

Neutrophils pretreated with cytochalasin were incubated for 2-30 min with [14C]-arachidonic acid in the presence or absence of drug and processed as escribed in Methods. I Mean value of duplicate determinations from a single experiment; all other values are means (?S.E.) derived from duplicate determinations from at least 3 different experiments. *p