Vol. 168, No. 1, 1990 April 16, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 194-199

PRIMING EFFECT OF 2,3-DIBENZYLBUTANE-1,4-DIOL (MAMMALIAN LIGNAN) ON SUPEROXIDE PRODUCTION IN HUMAN NEUTROPHILS

M. Morikawa,

Department 1432-1

M. Abe, Y. Yamauchi, and M. Tsuboi

M. Inoue,

of Pharmacology, Tokyo College of Pharmacy Horinouchi, Hachioji-shi, Tokyo 192-03, Japan

Received February 27, 1990 We investigated the effect of 2,3-dibenzylbutane-1,4dio (DBB), a mammalian lignan, on superoxide production and [Ca 3 +li mobilization in human neutrophils. DBB did not generate but enhanced the FMLP or A23187superoxide production by itself, induced superoxide production in a dose dependent manner. DBB did not influence the OAG-induced superoxide production. The priming effect of DBB was inhibited by W-7 or trifluoroperazine, but not by H-7 or staurosporine. And the priming effect of g!B was observed in the presence or absence of ex S+racellular Ca . DBB enhanced the low dose FMLP-induced [Ca Ii mobilization. These results suggest that the priming effect of DBB in human neutrophils may be caused by the activation of the calciumcalmodulin pathway but not the protein kinase pathway. B199O Rcademic Press,

Inc.

It

has

mammalian

been

reported

animals

diuretic

have

action

and factor

but

its

defense of

bacteria,

oxidase) of

system

in

important

is

found

of

in

human

in

the

2,3urine

mammalian

During

(4)

leading

to

host

phagocytosis

enzyme-complex

stimulated,

platelet-

unknown. roles

plasma-membrane-bound

action

Recently,

infection.

isolated

and virus

from

plants,

(7).

0006-291X/90$1.50 Copyright All rights

been

a

(the the

NADPH

production

radicals(S).

Some lignans (6)

(3).

is

microbial

neutrophils

superoxide

bacteria

some

against a

has

from

actions;

action

receptor

significance have

derived

a digitalis-like

antagonistic

(DBB)

physiological

Neutrophils

an

lignans

pharmacological B)( 11,

(PAF)

dibenzylbutane-1,4-diol

the

several

(prestegane

(enterolactone)(2) activating

that

0 1990 by Academic Press, Inc. of reproduction in any fom reserved.

194

have

effects

on cancer,

Vol.

168, No. 1, 1990

In

this

BIOCHEMICAL

study,

superoxide

we

AND BIOPHYSICAL

investigated

production

and

RESEARCH COMMUNICATIONS

the

[Ca2+li

effect

of

mobilization

DBB in

on

human

neutrophils.

AND METHODS

MATERIALS

Materials : DBB was provided from Tsumura. FMLP, Cytochrome C OAG, superoxide dismutase,chlorotetracycline (CTC) and (type VI), trifluoroperazine (TFP) were purchased from Sigma. Dextran and Ficoll-Paque were products of Pharmacia. A23187 was purchased from Hoechst. Fura 2-AM and HEPES were obtained from Dojin Laboratories. Hanks' balanced salt solution (HBSS) was purchased from MA Bioproducts. H-7 and W-7 were purchased from Seikagaku Kogyo. Staurosporine was obtained from Biomol Research Laboratories Inc. Fractionation of neutrophils : Neutrophils from healthy donors were isolated by the Dextrane-sedimentation, the centrifugation through Ficoll and the haemolysis of residual erythrocytes a described (8). The separated neutrophils were suspended (Ix10 I cells/ml)in HBSS (pH 7.4). Determination of superoxide production: NADPH-dependent superoxide production was determined by the superoxide dismutasesensitive rate of cytochrome c reduction as described by Cross et al.(g). Measurement of calcium mobilization: Neutrophils were loaded Fura 2-AM (3 uM)(IO) or CTC (100 uM)(II) for 40;pin at 37 C. The change of fluorescence was measured using a Ca + analyser (model CAF-100, Jasco). Intracelular free calcium concentration ([Ca2+]i) was calculated as described previously (IO). RESULTS AND DISC!DSSIOR

Effect ----

of

DBB on superoxide

By preincubating that

do not

of

untreated

cells.

has been

with

evoke

functions

human

using

superoxide

uM-100 superoxide

uM).

Fig.1

superoxide However, production.

is

shows

the

effect

but

production

These

in not

results 195

stimuli

DBB did

itself,

DBB did

to

to

as priming

and

(12,13).

of

enhance indicate

production

DBB on FMLP-

not

enhanced dose

the

relative

DBB on superoxide

production. by

concentration

augmented

of

of

low

by themselves,

referred

variety

effect

superoxide production

A23187-induced

are

a broad the

at

cell-functions

phenomenon

neutrophils.

A23187-induced

agonists

neutrophils

This

We investigated in

some

any observed

human

studied

production

generate the

dependent the that

or the

FMLP-

or

manner(I

OAG-induced DBB has

a

Vol.

168,

No.

BIOCHEMICAL

1, 1990

A

I

FMLP

45.6

AND

BIOPHYSICAL

A23187 320

nM

RESEARCH

COMMUNICATIONS

n&d

DBB

B

z i >

300

300

200

200

*

*

‘*

d 100

a 100 0 D

5

* LlilE

7

8 FMLP

-

Fig.

0

6

Control

A23187

M

6

7

8

(-loghl)

5

6

OAG

(-logM)

4

(-IogM)

DBB-pretreated

Effect of DBB on FMLP-, A23187- or OAG-induced --superoxide production --in human neutrophils A : Effect of DBB on FMLP-or A23187-induced superoxide production. B : Effect of DBB (100 uM) on the dose-response curve of FMLP, A23187 or OAG for superoxide prodcution in human platelets. Relative value to the response induced by 320 nM FMLP, 320 nM A23187 or 32 nM OAG in control. Mean+S.E. (n=4), * p