BIOLOGY

OF

REPRODUCTION

Stimulation

47,

1053-1058

(1992)

of Phospholipase

A2 by Xanthine

X.M.

Biology

Department,

WU,

M. SAWADA,

University

Oxidase

and

of Waterloo,

J.C.

in the Rat Corpus

Luteum1

CARLSON2

Waterloo,

Ontario,

Canada

N2L

3G1

ABSTRACT The

of the superoxide

ability

in microsomes in SOR prepared

from

prepared

formation

followed

from

control

or

nordthydroguaiaretic species

from

it was

and

mediate

acid,

the

cell.

highest

generated

(50R)

luteinized

rat

by xanthine

Treatment

ovaries.

by an increase

in PL.A,

prostaglandin

F,,, (PGF,,,)-treated

by superoxide

and

The

stimulation

in samples

activation,

PLA,

radical

which

dismutase

of PLA,

in which may

activity.

be involved

Stimulation and

activity was

cytosol

Prostaglandin

Recently,

F2a (PGF2a)

is known

to cause

appears to work through second steps in its mechanism of action

it

we

observed

that

was

metabolize

and

it

releases

substrate for leukotrienes)

arachidonic

acid,

intracellular messengers via the cyclooxygenase

pathways. The PLA2 enzymes

have

been

which

luteolytic

extensively

snake

venom.

Also,

a higher

molecular

phospholipases (e.g., 85-iOO Wa) located of mammalian cells has been described group of enzymes is ligand stimulated,

activity

chidonic

acid, and

has been

found

it was associated tion. The purpose

with

the

PGF2a trolling

luteum (CL) contains The concentrations

reproductive [6, 7]. luteal

It

cycle

been

(SOD)

implicated

they

are

class of

in the cytoplasm [4,5]. The latter selective for aracalcium-

sensitive

Superoxide SOR are

radicals metabolized

to H202,

and

reactive

in luteal

regression.

Xanthine oxidase, SOD, (NDGA), bromophenacyl

catalase, bromide,

dylcholine (Type ical Co. (St. Louis, F-bOO)

In experiments

to that transient

oxygen speof proges-

caused by increase

PGF

in lu-

PGF2a treatment in PLA2 activity,

in and

secreto study

METHODS

nordihydroguaiaretic acid and egg L-a-phosphati-

V-E) were purchased MO). Vitamin E (d-a

a gift

from

Henkel

from

MN),

from Boehringer radiolabeled phos-

i-14C]arachidonyl was

ChemCovitol

(Minneapolis,

purchased PQ), and

(1 -stearoyl-2-[ 58, mCi/mmol)

from Sigma tocopherol,

Corp.

(R24571) was Canada Ltd. (Laval,

phosphati-

Amersham

(Arlington

IL).

Immature rats were (Equinex, 50

(24-28 days old) Wistar-derived, superovulated by s.c. injection Ayerst Laboratories, Montreal,

LU of hCG

have

experiments, p.g of PGF2a

with

Orangeville,

ously 1053

Chemical

on

Co.)

rats were Tromethamine,

Day

6 after

white female of 50 LU of eCG PQ) followed by

65 also

hCG

h later.

In

injected with Tuco Products

administration

some 500

Co., to in-

regression.

Samples

Samples

Canada

(Sigma superovulated (Dinoprost ON)

luteal

Ovarian Research Council of Canada. of Waterloo, Waterloo, Ontario

may

Animals

duce Accepted August 3, 1992. Received April 7, 1992. ‘This work was supported by the Medical ‘Correspondence: Dr.J.C. Carlson, University N2L 3G1. FAX: (519) 746-0614.

was

calmidazolium Mannheim, dylcholine; Heights,

in concan reg-

species

ions,

11,0,

PLA2 in the CL by using xanthine to see if reactive oxygen species this enzyme during luteolysis.

AND

phatidylcholine

(SOR) are presby superoxide

oxygen

of calcium and/or

with the decrease in progesterone of the current investigation was

MATERIALS

to U-I and

involved vitamins

addition the SOR

ChemicaLs

high levels of antioxof these vitamins vary

is possible that they are function, since antioxidant

ulate oxygen radicals. ent in all aerobic cells. dismutase

and

E and oxygen

generation of reactive causes rapid inhibition

the mechanism activating oxidase to generate SOR are involved in regulating

dependant translocation to membrane surfaces during activation. The mechanism responsible for stimulation of P[A2 in the luteal cell is unknown. The corpus idant vitamins.

vitamin

reactive

formation in vivo shortly after [9]. This rise preceded the increase

[3]. They

to undergo

burst

microsomes

in

SOR

rats

as the

mass

that

examined

process.

teal

are generally believed to be calcium dependent and are found in different forms including a class of secreted, low molecular mass (12-18 Wa) enzymes that appear in bee and

similar

to remove the

upon

indicate

was

in a rapid

antioxidants,

H,02

and

dependent results

the

and

terone synthesis in a manner similar [8]. Recently we observed a rapid,

(e.g., prostaglandins, or the lipoxygenase

studied

by

SOR

(PLA2)

resulted

related

dose

inhibited

was These

oxidase

dispersed rat luteal cells, cies by xanthine oxidase

luteolysis.

serves

oxidase

to membranes.

A,

phospholipase

xanthine was

which

increases as progesterone levels fall in the blood during corpus luteum regression in pregnant and pseudopregnant rats [1]. PLA2 is the rate-limiting enzyme in PGF2 synthesis [2],

activity

Activation

messengers, are unknown.

A2 (PLA2)

phospholipase

of PLA,

with

rats.

INTRODUCTION

Although the detailed

to activate

catalase,

by xanthine

added

in the

oxidase

of microsomes

described

of crude [1].

microsomes Briefly,

were the

ovaries

obtained of 2-4

as previrats

were

WU

1054

homogenized

and

supernatant

was

for 30 mm. crosomes)

study. were from

the

23000

10 mm.

The

at 23000

x g

(cytosol). buffer and

and

supernatant

The pellet, recentrifuged were

Protein

procedure

of ovarian tissue They were prepared

x g supernatant by centrifuging to produce a pellet (membranes)

pernatant Tris-HCI the

X g for

recentrifuged

additional preparations in some experiments.

g for 60 mm

activation.

at 2500 and

The supernatant of the second spin (crude miwas used for most of the experiments in this

However, also used

X

centrifuged removed

at 100 000 and su-

was

used

in the

determined

study

of

by the

PLA2 50

[10].

beled phosphatidylcholine as previously described [1]. However, the procedure was modified as follows. Samples (0.1 mg of protein in 0.5 ml of buffer) containing crude (23 000

microsomes the l

100

X g spin) or membranes

x g spin

000

of liposomes

(5.0

tg

were

were

separated viously

extracted

with

by thin layer [1]. To examine

or cytosol

preincubated

of egg

(4#{176}C) with

phosphandylcholine

0.3 pg of radiolabeled phosphatidylcholine, for 15 mm followed by various treatments figure legends. The treated samples were at 40#{176}C for 60 mm with 1.0 mM CaCl2. After lipids

and

44000 cpm) as indicated in then incubated incubation, the

chloroform:methanol

chromatography the effect of free

(2:1)

hibiting PLA2 activity, U; of catalase, 1 mg;

To

Spin levels

measured tein

Resonance

(ESR)

SOR

formation

determine

ments,

the optimum and of vitamin

of

this

radical

as described

[9]. Tiron

during crude

using

in

was

vitro

100

were

100 pg of sample

pro-

(1,2-dihydroxybenzene-3,5-di-

times

that

corresponded

to those

for

the

at PLA2

assay.

Stat is-tics Each separate replicate.

experiment times using Significance

in this

study

was

repeated

separate microsome was determined

of variance (ANOVA) followed by mining differences among groups Range test for within group way ANOVA was performed

three

samples by one-way

Tukey’s test or Duncan’s

differences. for comparing

or four for each analysis

for deterMultiple

In addition, the effect

20

30

oxidase

in

samples

containing

cytosol

and

mem-

branes.

RESULTS Treatment some) with

of the xanthine

23 000 X g supernatant oxidase caused a rapid

twoof xan-

(crude burst

microof SOR

and stimulated a marked increase in PLA2 activity, as indicated by the percent formation of 14C-arachidonic acid (Fig. 1). SOR levels peaked at 4-5 mm and declined to basal before xanthine

there was a significant rise in PLA2 activity. oxidase-simulated PLA2 activity was dose de-

enhance this Pretreatment

response. of the

crude

doses of xanthine constant, relative and catalase also

oxidase, inhibition inhibited

activity (p