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