Vol. 78, No. 4, 1977
BIOCHEMICAL
INHIBITION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF PROSTAGLANDIN SYNTHESIS BY LYSOLECITHIN* W. Thomas Shier
Cell
Biology Laboratory, The Salk Institute Post Office Box 1809, San Diego,
Received
August
for Biological California 92112
Studies,
23,1977 SUMMARY
Exogenous lysolecithin inhibits prostaglandin E2 synthesis from arachidonic acid in bovine seminal vesicle microsomes at plausible physiological levels (lysolecithin-to-protein ratios b 0.03 [w/w]) by inhibiting fatty acid cycle-oxygenase activity. Structurally defined lysolecithins with varying fatty acid chain length exhibit varying effectiveness as inhibitors. Addition of equimolar quantities of free fatty acid lowers the lysolecithin concetration required for inhibition. Exogenous lysolecithin inhibits unstimulated and thrombin-stimulated prostaglandin E2 synthesis from endogenous substrate in SVBalb/3T3 cells. Serum treatment of SVBalb/3T3 cells, which generates endogenous lysolecithin and free fatty acids, decreases the efficiency of conversion of free arachidonic acid to prostaglandins. These results suggest a possible role for the products of phospholipase A2 action in the regulation of prostaglandin synthesis. This
laboratory
of the lipid
has been
composition
A2 may play
a role
investigating
of membranes
in the regulation
particular
guanylate
cyclase
(l-3).
can modify
guanylate
cyclase
activity
activity
is
PGs (4,5) activate
the first
including
to be expressed fatty
acids
via
is
*
so that
This Health
work Service
Abbreviations:
products
guanylate guanylate
prolonged only that
both
is
enzymes,
limiting
step
indeed,
the (8).
2) Lysolecithin
cyclase
at 1 pM [6])
of PG effects
at lower
activation
than
(1,2)
A2 action
presumably
A2
the biosynthesis have been shown
majority
of phospholipase cyclase,
1) Phospholipase in
PG endoperoxides
in A2 activity
appear and free
on membrane
by surfactant levels
of to
effects.
(half-maximal
(half-maximal However, in 3T3 membranes is at 80 pM [l]) or free fatty acids. are unstable with a half-life of approximately 5 min in buffer
be achieved
conceivable
rate
modification phospholipase
of phospholipase
by two mechanisms.
nucleotides
activate
in platelets
stimulation endoperoxides (101, could
cyclic
activate
PG endoperoxides stimulation
(6,7);
the major
(9),
phospholipids,
of membrane-associated
and probably cyclase
that
activatable
The products
PG endoperoxides.
guanylate
the possibility
by endogenous
of guanylate
by expenditure
of large
PG endoperoxides
Copyright 0 I977 by Academic Prexr, Inc. All righfs oj reproduction in any form reserved.
cyclase amounts
by PG endoperosides of PG precursors.
and lysolecithin
was supported by the Theodore Grants CA16123 and CA14195 DME, Dulbecco-modified SE, standard error.
lysolecithin
plus
Gildred Foundation from the National
Eagle's
medium;
free
It fatty
and U.S. Public Cancer Institute.
PG, prostaglandin;
1168 ISSN
0006-291
x
Vol. 78, No. 4, 1977
BIOCHEMICAL
acids
in the activation
could
former
be involved
being
being
responsible
responsible
for
activation
of endogenous by-product cyclase
the PG precursors
further
be averted
shorten
synthesis
(i.e.
guanylate
cyclase.
of regulatory synthesis free
this
fatty
acid
mechanism
acids
line
vesicle
the effects microsomes
PG synthesis MATERIALS
inhibited
the biochemical
in order
40 min.
simultaneously
at concentrations
SVBalb/3T3.
inhibit
produced
acids
of
be produced
expenditure
fatty
investigated
by determining
seminal
cell
fatty
and free
cycle-oxygenase)
We have
in bovine
The unnecessary
time.
lysolecithin
specificity,
1 UM PGG2 for
acids
assuming
activation
(1) would
fatty
latter
For example,
maximal
to maintain
the
and the
no phospholipid half
preparations by free
with
activation
A2 with
required
cyclase,
activation.
to induce
cyclase
if
mouse fibroblast that
more prolonged
in 3T3 membrane
of guanylate
could
of guanylate
phospholipase
to provide
RESEARCH COMMUNICATIONS
short-duration
lysolecithin
Activation might
rapid,
slower
sufficient guanylate
for
AND BIOPHWCAL
of PG precursors PG endoperoxide that
basis
for
of lysolecithin
activate this
type
on PG
and in a well-characterized
Robak --et al. (11) have demonstrated in microsomal preparations.
AND METHODS
Prostaglandins E2 and F2e, were the gifts of Dr. John Pike, The Upjohn co.. , Kalamazoo, Mich. Highly purified (12) direct lytic factor from the venom of African Ringhals Cobra (Haemachatus haemachates) was the gift of Dr. David Eaker, University of Up&ala. Bovine thrombin (topical grade) was obtained from Parke-Davis. [5,6,8,9,11,12,14,15(n)-3H]-Arachidonic acid (80 Ci/mmole) was obtained from Amersham/Searle. Palmitoyl[choline methyl14C]-L-cL-lysolecithin was prepared from dipalmitoyl[choline methyl-14C]lecithin (New England Nuclear) by treatment with phospholipase A2 (Crotalus adamanteus venom, Worthington) according to the method of Wells and Hanahan (13). Unless otherwise indicated, all other biochemicals were obtained from Sigma Chemical co. Protein was determined by tryptophan fluorescence (14) using bovine serum albumin as standard. Bovine seminal vesicle microsomes were prepared from frozen tissue (Pel-Freeze Biologicals, Rogers, Ark.) by the method of Flower -et -*al SVBalb/3T3 cells were obtained from Dr. M. Vogt and (15). maintained in a humidified 10% CO2 atmosphere in DME containing 10% calf serum (Irvine Scientific Sales Co.) using 0.05% trypsin for subculturing the cells. RESULTS &hibition
of prostaglandin
Exogenous graphically
egg yolk
synthesis
lysolecithin
identifiable
in bovine
strongly
PGE2 from
seminal
inhibits
arachidonic
vesicle
the synthesis acid
in bovine
microsomes. of chromatoseminal
vesicle
microsomes (Fig. IA). Arachidonic acid conversion products that cochromatograph with PGE2, with PGF2, and with PGD2 (not shown), are all inhibited by similar concentrations of lysolecithin. Concomitant inhibition of the synthesis of these three PGs suggests that fatty acid cycle-oxygenase is
inhibited
these
products.
by lysolecithin, Indomethacin
since
it
is
and lysolecithin
1169
involved inhibit
in
the synthesis
of all
PG synthesis
indepen-
of
Vol. 78, No. 4, 1977
RATIO
01
BIOCHEMICAL
OF EXOGENOUS
T O ENZYME
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LYSOLECITHIN
PROTEIN
(W/W
02
x100)
INDOMETHACIN
CONC.
t/d)
of egg yolk lysolecithin (A) and of equimolar amounts of Fig. 1. Effect lysolecithin and sodium oleate (B) on the synthesis of prostaglandin E2 (-0-) and F-type prostaglandin (-O-) ri SE from tritiated exogenous [3H]arachidonic acid in bovine seminal vesicle microsomes prepared and assayed in triplicate by the radiometric method of Flower --et al. (15) using 3 mg/ml enzyme protein preincubated 1 min with added lipids and incubated 3 min at 30' following initiation of the assay with 100 uM [3H]-arachidonic acid. The percent conversion of substrate to PGs was determined by thin layer chromatography of extracted lipids using the solvent system ethyl acetate: acetone:acetic acid (90:10:1), followed by liquid scintillation counting of the radioactivity migrating with authentic PGE2, PGF2, and arachidonic acid internal standards and the radioactivity in the remainder of the chromatogram. Fig. 2. Effect of exogenous egg yolk lysolecithin (-•-) and lecithin at lipid-to-protein ratios of 0.17 (w/w) in triplicate on the inhibition indomethacin of prostaglandin E2 synthesis from t3H]-arachidonic acid bovine seminal vesicle microsomes assayed as described in Fig. 1.
dent
of each other
mechanisms. (Table
l),
with
at lower
lecithins
(egg yolk
of PGE2 synthesis
suggesting defined containing
concentrations. lysolecithin) than
result
from more effective
result
of lower
critical
lysolecithin
A lower
of mixed
for
they
shorter
were
fatty
acid
for
half-maximal
inhibitory
carbon
chains
lyso-
inhibition The
lysolecithins. lysolecithins
to microsomal
produced
1170
weight
all
of mixed
any of the defined
of lipid is
by different
concentration
temperatures
in membranes
act
lysolecithins
and low molecular
transfer micellar
that
was required
was required
effectiveness
Since
2),
of structurally
the lysolecithins
inhibiting
increased
(see Fig.
A series
(-0-) by by
membranes
may as a
(16). predominantly
by the action
Vol. 78, No. 4, 1977
Table
1.
BIOCHEMICAL
Inhibition
microsomes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of prostaglandin
by structurally
E2 synthesis
defined
in bovine
seminal
vesicle
L-a-lysolecithins.
-______ Number of carbons L-a-lysolecithin
in
fatty
Lysolecithin-to-protein
acid
causing
ratio
50% inhibition
of PGE2 a
synthesis -___ Lauroyl Myristoyl Palmitoyl
-
Stearoyl -
12 14 16
0.16 0.27
18
0.46
0.40 -
a Assayed
as described
in Fig.
1.
of endogenous
phospholipase
A2 on lecithin,
produced
each molecule
of lysolecithin.
with
PGE2 synthesis lower Inhibition
cells
into
the
lipids
protein
arachidonic
acid
stimulated
being
induced
a similar
to PGF.
examined
radioactivity
the medium
(i.e.
PGs) indicates As observed
from
in
lipids
this
of free acid
lysolecithin
The
in growing
thrombin3).
at lipid-
PGE2 synthesis
from
and lysolecithin-
The mechanism
by exogenous
of
lysolecithin
is
Exogenous lysolecithin of conversion of free arachidonic
arachidonic into
acid
to PGE2 and release
the medium are
to total
by Hong and Levine
cells.
laboratory.
free
of conversion
prostaglandin
cells with serum activates higher than does treatment with thrombin. conversion of free [3H]-arachidonic
of
of [3H]-arachidonic
inhibited
(Fig.
the efficiency
arachidonic
SVBalb/3T3
by both
activity in
in PGE2 relative relative
is
an equimolar
was determined
Exogenous
activity
the efficiency
acid
at significantly
added with
incorporation and higher
deacylating
conversion
free
cell
further
variation
Since
the unconverted
in cultured
(17,L8). (w/w)
deacylating
of endogenous
currently
is
fatty
inhibition
occurs
on PGE2 synthesis
by biosynthetic
produced
endogenous
activation
E2 synthesis
of 0.015
of free
1B).
of the cell
ratios
microsomes
when lysolecithin
lysolecithin labelled
a molecule Half-maximal
vesicle
(see Fig.
of prostaglandin
to-cell
the
oleate
of exogenous
SVBalb/3T3
acid
seminal
concentrations
of sodium
effect acid
in bovine
lysolecithin
amount
(w/w)
synthetase (17,19),
fatty
of free
acid
competing
radioactivity
arachidonic
of
processes, acid
in to
activity.
treatment
of transformed
Balb/3T3
levels of endogenous deacylating activity Significantly decreased efficiency of acid
1171
generated
in radiolabelled
SVBalb/3T3
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
30 I I IO Q-
1
r-t.---.rs-.f-T.-; 0.03 0.1 0.2
--=A 0 RATIO
OF EXOGENOUS
0.5
I
LYSOLECITHIN (W/W
235 T O CELL
PROTEIN
x 100)
Fig. 3. Effect of exogenous lysolecithin on prostaglandin E2 synthesis in cultured SVBalb/3T3 cells determined by the method of Hong and Levine (17,18). Triplicate cultures (2 x lo5 cells plated in 3.2 cm diameter dishes) were radiolabelled by incorporation from 1 pCi of [3H]-arachidonic acid during 24 hr in 2 ml DME containing 10% calf serum. About 60% of the added radioactivity is taken up by the cells and about 98% is converted to phospholipids and triglycerides. The cells were washed on the dishes 3 times with 2 ml DME, and the amount _+ SE of PGE2 (-0-) and total free fatty acid (-O-) radioactivity released in 30 min at 37O into 0.6 ml of DME containing the indicated concentrations of lysolecithin was determined by extracting and chromatographing lipids released into the medium using the method described in Fig. 1, and expressed as a percentage of the radioactivity incorporated into the cells. Under these conditions Q 92% of free arachidonic acid, 90% of PGE2 and 80% of PGF radioactivity was released into the culture medium. The amount of exogenous lysolecithin absorbed by the cells during 30 min at each concentration was estimated in parallel cultures by monitoring the percentage of [choline methyl-14C]-lysolecithin absorbed. The release f SE of PGE2 radioactivity during the next 30 min into an additional 0.6 ml DME containing bovine thrombin (10 pg/ml) (-El-) was determined in the same manner. Cell protein was 380 I-lg per dish.
cells
to PGE2 was observed
concentrations
b
5%.
tase enzyme system,
This
since
(see Fig. result
4) following is
treatment
not
with
treatment
due to saturation direct
lytic
with
calf
serum at
of the PG synthe-
factor
from African
Ringhals Cobra (30 pg/ml in DME) in parallel cultures stimulated the synthesis of 1.6 times as much PGE2 as the maximum stimulated by serum and thrombin (i.e.
at 5% serum). DISCUSSION A normal
be calculated This
amount
microsomes
basal from
lysolecithin-to-protein the
of lysolecithin (Fig.
data
ratio
of
Ray et al.
is
not
1) or in SVBalb/3T3
inhibitory cells
1172
(19)
equal for
rat
in either (Fig.
3).
to 0.0123 liver bovine
(w/w)
plasma
seminal
A lecithin-to-protein
can
membranes. vesicle
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.Ii O-?i-
0.1
0.2
CONC.
OF
0.5
I
2
CALF
SERUM
5
IO
(%I
Fig. 4. Effect of stimulation of SVBalb/3T3 cells for 30 min by a range of -__ concentrations of calf serum on endogenous phospholipase activity3+ SE (-a-), and on the efficiency of conversion ? SE of the released [ H]arachidonic acid tc PGE2 during stimulation by serum alone (-U-) or in paralle cultures by serum with thrombin (10 pg/ml) (-0-), determined by the methods described in Fig. 3. The efficiency of conversion of free arachidonic acid to Serum-activated F-type PG exhibited similar changes with serum concentration. endogenous deacylating activity in SVBalb/3T3 cells generates endogenous lysolecithin as well as free fatty acids (22).
ratio
of 0.24
(w/w)
can be calculated
of Ray et al.
(19);
the lysolecithin-to-protein
inhibition
of PGE2 synthesis
Fig. 1B) would of the Lecithin Treatment labelled
cell
lipids
in about
1 hr
(5.8%
ments;
a similar
Il71). inhibits
Because
modify provide
acids
membranes that
vesicle
5% serum stimulates
that
would
30 min in Fig.
achieve
fatty
affect
acid
other
elevated
of
these
biochemical
levels enzymes basis
degree
enzymes, in membranes
in a coordinate for
(i.e.
a proposed-(l)
of prostaglandin synthesis by lysolecithin a possible explanation for the prostaglandin
by Manku tricyclic
and Horrobin
(20)
antidepressant
with
certain
local
and methylxanthine
1173
0.03,
of
of hydrolysis
2 hr in some experiof Hong and Levine
at which
activity
membrane-associated
lysolecithin
50%
hydrolysis of 12.5% ratio of 0.24 (w/w).
from the results
cycle-oxygenase
the data
the hydrolysis
this
ratios
causes
microsomes
4, and up to 25% in
can be calculated
from
lysolecithin
(Fig.
1) are
including would
similar
nucleotide be expected
fashion.
These
mechanism
regulation of membrane-associated enzymes by alteration lipid composition in response to external stimuli.
Inhibition offers
arrhythmic,
with
at a rate
value
additional
observed
seminal
the lysolecithin-to-protein
the activities
coordinate membrane
liver ratio
in bovine
cells
in
microsomal that (l),
rat
be produced by phospholipase A2 catalyzed in membranes with a lecithin-to-protein
of SVBalb/3T3
to those cyclases
for
and free antagonist anesthetic, compounds.
for
to
studies the
of cell fatty effects antiCompounds
Vol. 78, No. 4, 1977
of these
types
have been observed
A:lysolecithin elevated lower
acyltransferase levels
lysolecithin
BIOCHEMICAL
of lysolecithin may exhibit
PG levels
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be effective
activity
tissues
Acknowledgment - I gratefully Mr. J. T. Trotter.
from several
in treated
PG synthesis
inhibitors
tissues
without
and apparent
sources (21).
inhibiting
PG antagonist
acknowledge
the
of acyl
technical
coenzyme
and they
Elevated
cause
levels
degradation,
of
causing
effects. assistance
of
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Shier, W. T., Baldwin, J. H., Nilsen-Hamilton, M., Hamilton, R. and Thanassi, N. M. (1976) Proc. Natl. Acad. Sci. USA, 73, 1586-1590. Shier, W. T. and Trotter, J. T. (1976) Fed. Proc., 25, 1731. Shier, W. T. and Trotter, J. T. (1976) FEBS Lett., 62, 165-168. Kunze, H. and Vogt, W. (1971) Ann. N.Y. Acad. Sci., 180, 123-125. Samuelsson, B. (1972) Fed. Proc., 2, 1442-1450. Glass, D. B., Gerrard, J. M., Townsend, D., Carr, D. W., White, J. G. and Goldberg, N. D. (1977) .i. Cyclic Nucl. Res., 1, 37-44. Gorman, R. R., Hamberg, M. and Samuelsson, B. (1975) J. Biol. Chem., 25J, 6460-6463. Kuehl, F. A. (1974) Prostaglandins, 5, 325-340. Asakawa, T., Scheinbaum, I. and Ho, R. .I. (1976) Biochem. Biophys. Res. Commun., 12. 141-148. Hamberg, M., Svensson, J., Wakabayashi, T. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. USA, 71, 345-349. Robak, J., Dembinska-Kiec, A. and Gryglewski, R. (1975) Biochem. Pharm., 24, 2057-2060. Fryklund, L. and Eaker, D. (1973) Biochemistry, 12, 661-667. Wells, M. A. and Hanahan, D. J. (1969) Biochemistry, 8, 414-424. J. and Martin, D. B. (1972) J. Biol. Chem., --) 247 Carter, J. R., Avruch, 2682-2688. Flower, R. J., Cheung, H. S. and Cushman, D. W. (1973) Prostaglandins, 6, 325-341. Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta, 415, 29-79. Hong, S. L. and Levine, L. (1976) Proc. Natl. Acad. Sci. USA, 73, 1730-1734. Hong, S. L. and Levine, L. (1976) J. Biol. Chem., 251, 5814-5816. Ray, T. K., Skipski, V. P., Barclay, M., Essner, E. and Archibald, F.M. (1969) J. Biol. Chem., 2/&, 5528-5536. Manku, M. S. and Horrobin, D. F. (1976) Prostaglandins, 2, 789-801. Shier, W. T. (1977) Biochem. Biophys. Res. Commun., 2, 186-193. Shier, W. T., manuscript in preparation.
1174
BIOCHEMICAL
INHIBITION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF PROSTAGLANDIN SYNTHESIS BY LYSOLECITHIN* W. Thomas Shier
Cell
Biology Laboratory, The Salk Institute Post Office Box 1809, San Diego,
Received
August
for Biological California 92112
Studies,
23,1977 SUMMARY
Exogenous lysolecithin inhibits prostaglandin E2 synthesis from arachidonic acid in bovine seminal vesicle microsomes at plausible physiological levels (lysolecithin-to-protein ratios b 0.03 [w/w]) by inhibiting fatty acid cycle-oxygenase activity. Structurally defined lysolecithins with varying fatty acid chain length exhibit varying effectiveness as inhibitors. Addition of equimolar quantities of free fatty acid lowers the lysolecithin concetration required for inhibition. Exogenous lysolecithin inhibits unstimulated and thrombin-stimulated prostaglandin E2 synthesis from endogenous substrate in SVBalb/3T3 cells. Serum treatment of SVBalb/3T3 cells, which generates endogenous lysolecithin and free fatty acids, decreases the efficiency of conversion of free arachidonic acid to prostaglandins. These results suggest a possible role for the products of phospholipase A2 action in the regulation of prostaglandin synthesis. This
laboratory
of the lipid
has been
composition
A2 may play
a role
investigating
of membranes
in the regulation
particular
guanylate
cyclase
(l-3).
can modify
guanylate
cyclase
activity
activity
is
PGs (4,5) activate
the first
including
to be expressed fatty
acids
via
is
*
so that
This Health
work Service
Abbreviations:
products
guanylate guanylate
prolonged only that
both
is
enzymes,
limiting
step
indeed,
the (8).
2) Lysolecithin
cyclase
at 1 pM [6])
of PG effects
at lower
activation
than
(1,2)
A2 action
presumably
A2
the biosynthesis have been shown
majority
of phospholipase cyclase,
1) Phospholipase in
PG endoperoxides
in A2 activity
appear and free
on membrane
by surfactant levels
of to
effects.
(half-maximal
(half-maximal However, in 3T3 membranes is at 80 pM [l]) or free fatty acids. are unstable with a half-life of approximately 5 min in buffer
be achieved
conceivable
rate
modification phospholipase
of phospholipase
by two mechanisms.
nucleotides
activate
in platelets
stimulation endoperoxides (101, could
cyclic
activate
PG endoperoxides stimulation
(6,7);
the major
(9),
phospholipids,
of membrane-associated
and probably cyclase
that
activatable
The products
PG endoperoxides.
guanylate
the possibility
by endogenous
of guanylate
by expenditure
of large
PG endoperoxides
Copyright 0 I977 by Academic Prexr, Inc. All righfs oj reproduction in any form reserved.
cyclase amounts
by PG endoperosides of PG precursors.
and lysolecithin
was supported by the Theodore Grants CA16123 and CA14195 DME, Dulbecco-modified SE, standard error.
lysolecithin
plus
Gildred Foundation from the National
Eagle's
medium;
free
It fatty
and U.S. Public Cancer Institute.
PG, prostaglandin;
1168 ISSN
0006-291
x
Vol. 78, No. 4, 1977
BIOCHEMICAL
acids
in the activation
could
former
be involved
being
being
responsible
responsible
for
activation
of endogenous by-product cyclase
the PG precursors
further
be averted
shorten
synthesis
(i.e.
guanylate
cyclase.
of regulatory synthesis free
this
fatty
acid
mechanism
acids
line
vesicle
the effects microsomes
PG synthesis MATERIALS
inhibited
the biochemical
in order
40 min.
simultaneously
at concentrations
SVBalb/3T3.
inhibit
produced
acids
of
be produced
expenditure
fatty
investigated
by determining
seminal
cell
fatty
and free
cycle-oxygenase)
We have
in bovine
The unnecessary
time.
lysolecithin
specificity,
1 UM PGG2 for
acids
assuming
activation
(1) would
fatty
latter
For example,
maximal
to maintain
the
and the
no phospholipid half
preparations by free
with
activation
A2 with
required
cyclase,
activation.
to induce
cyclase
if
mouse fibroblast that
more prolonged
in 3T3 membrane
of guanylate
could
of guanylate
phospholipase
to provide
RESEARCH COMMUNICATIONS
short-duration
lysolecithin
Activation might
rapid,
slower
sufficient guanylate
for
AND BIOPHWCAL
of PG precursors PG endoperoxide that
basis
for
of lysolecithin
activate this
type
on PG
and in a well-characterized
Robak --et al. (11) have demonstrated in microsomal preparations.
AND METHODS
Prostaglandins E2 and F2e, were the gifts of Dr. John Pike, The Upjohn co.. , Kalamazoo, Mich. Highly purified (12) direct lytic factor from the venom of African Ringhals Cobra (Haemachatus haemachates) was the gift of Dr. David Eaker, University of Up&ala. Bovine thrombin (topical grade) was obtained from Parke-Davis. [5,6,8,9,11,12,14,15(n)-3H]-Arachidonic acid (80 Ci/mmole) was obtained from Amersham/Searle. Palmitoyl[choline methyl14C]-L-cL-lysolecithin was prepared from dipalmitoyl[choline methyl-14C]lecithin (New England Nuclear) by treatment with phospholipase A2 (Crotalus adamanteus venom, Worthington) according to the method of Wells and Hanahan (13). Unless otherwise indicated, all other biochemicals were obtained from Sigma Chemical co. Protein was determined by tryptophan fluorescence (14) using bovine serum albumin as standard. Bovine seminal vesicle microsomes were prepared from frozen tissue (Pel-Freeze Biologicals, Rogers, Ark.) by the method of Flower -et -*al SVBalb/3T3 cells were obtained from Dr. M. Vogt and (15). maintained in a humidified 10% CO2 atmosphere in DME containing 10% calf serum (Irvine Scientific Sales Co.) using 0.05% trypsin for subculturing the cells. RESULTS &hibition
of prostaglandin
Exogenous graphically
egg yolk
synthesis
lysolecithin
identifiable
in bovine
strongly
PGE2 from
seminal
inhibits
arachidonic
vesicle
the synthesis acid
in bovine
microsomes. of chromatoseminal
vesicle
microsomes (Fig. IA). Arachidonic acid conversion products that cochromatograph with PGE2, with PGF2, and with PGD2 (not shown), are all inhibited by similar concentrations of lysolecithin. Concomitant inhibition of the synthesis of these three PGs suggests that fatty acid cycle-oxygenase is
inhibited
these
products.
by lysolecithin, Indomethacin
since
it
is
and lysolecithin
1169
involved inhibit
in
the synthesis
of all
PG synthesis
indepen-
of
Vol. 78, No. 4, 1977
RATIO
01
BIOCHEMICAL
OF EXOGENOUS
T O ENZYME
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LYSOLECITHIN
PROTEIN
(W/W
02
x100)
INDOMETHACIN
CONC.
t/d)
of egg yolk lysolecithin (A) and of equimolar amounts of Fig. 1. Effect lysolecithin and sodium oleate (B) on the synthesis of prostaglandin E2 (-0-) and F-type prostaglandin (-O-) ri SE from tritiated exogenous [3H]arachidonic acid in bovine seminal vesicle microsomes prepared and assayed in triplicate by the radiometric method of Flower --et al. (15) using 3 mg/ml enzyme protein preincubated 1 min with added lipids and incubated 3 min at 30' following initiation of the assay with 100 uM [3H]-arachidonic acid. The percent conversion of substrate to PGs was determined by thin layer chromatography of extracted lipids using the solvent system ethyl acetate: acetone:acetic acid (90:10:1), followed by liquid scintillation counting of the radioactivity migrating with authentic PGE2, PGF2, and arachidonic acid internal standards and the radioactivity in the remainder of the chromatogram. Fig. 2. Effect of exogenous egg yolk lysolecithin (-•-) and lecithin at lipid-to-protein ratios of 0.17 (w/w) in triplicate on the inhibition indomethacin of prostaglandin E2 synthesis from t3H]-arachidonic acid bovine seminal vesicle microsomes assayed as described in Fig. 1.
dent
of each other
mechanisms. (Table
l),
with
at lower
lecithins
(egg yolk
of PGE2 synthesis
suggesting defined containing
concentrations. lysolecithin) than
result
from more effective
result
of lower
critical
lysolecithin
A lower
of mixed
for
they
shorter
were
fatty
acid
for
half-maximal
inhibitory
carbon
chains
lyso-
inhibition The
lysolecithins. lysolecithins
to microsomal
produced
1170
weight
all
of mixed
any of the defined
of lipid is
by different
concentration
temperatures
in membranes
act
lysolecithins
and low molecular
transfer micellar
that
was required
was required
effectiveness
Since
2),
of structurally
the lysolecithins
inhibiting
increased
(see Fig.
A series
(-0-) by by
membranes
may as a
(16). predominantly
by the action
Vol. 78, No. 4, 1977
Table
1.
BIOCHEMICAL
Inhibition
microsomes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of prostaglandin
by structurally
E2 synthesis
defined
in bovine
seminal
vesicle
L-a-lysolecithins.
-______ Number of carbons L-a-lysolecithin
in
fatty
Lysolecithin-to-protein
acid
causing
ratio
50% inhibition
of PGE2 a
synthesis -___ Lauroyl Myristoyl Palmitoyl
-
Stearoyl -
12 14 16
0.16 0.27
18
0.46
0.40 -
a Assayed
as described
in Fig.
1.
of endogenous
phospholipase
A2 on lecithin,
produced
each molecule
of lysolecithin.
with
PGE2 synthesis lower Inhibition
cells
into
the
lipids
protein
arachidonic
acid
stimulated
being
induced
a similar
to PGF.
examined
radioactivity
the medium
(i.e.
PGs) indicates As observed
from
in
lipids
this
of free acid
lysolecithin
The
in growing
thrombin3).
at lipid-
PGE2 synthesis
from
and lysolecithin-
The mechanism
by exogenous
of
lysolecithin
is
Exogenous lysolecithin of conversion of free arachidonic
arachidonic into
acid
to PGE2 and release
the medium are
to total
by Hong and Levine
cells.
laboratory.
free
of conversion
prostaglandin
cells with serum activates higher than does treatment with thrombin. conversion of free [3H]-arachidonic
of
of [3H]-arachidonic
inhibited
(Fig.
the efficiency
arachidonic
SVBalb/3T3
by both
activity in
in PGE2 relative relative
is
an equimolar
was determined
Exogenous
activity
the efficiency
acid
at significantly
added with
incorporation and higher
deacylating
conversion
free
cell
further
variation
Since
the unconverted
in cultured
(17,L8). (w/w)
deacylating
of endogenous
currently
is
fatty
inhibition
occurs
on PGE2 synthesis
by biosynthetic
produced
endogenous
activation
E2 synthesis
of 0.015
of free
1B).
of the cell
ratios
microsomes
when lysolecithin
lysolecithin labelled
a molecule Half-maximal
vesicle
(see Fig.
of prostaglandin
to-cell
the
oleate
of exogenous
SVBalb/3T3
acid
seminal
concentrations
of sodium
effect acid
in bovine
lysolecithin
amount
(w/w)
synthetase (17,19),
fatty
of free
acid
competing
radioactivity
arachidonic
of
processes, acid
in to
activity.
treatment
of transformed
Balb/3T3
levels of endogenous deacylating activity Significantly decreased efficiency of acid
1171
generated
in radiolabelled
SVBalb/3T3
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
30 I I IO Q-
1
r-t.---.rs-.f-T.-; 0.03 0.1 0.2
--=A 0 RATIO
OF EXOGENOUS
0.5
I
LYSOLECITHIN (W/W
235 T O CELL
PROTEIN
x 100)
Fig. 3. Effect of exogenous lysolecithin on prostaglandin E2 synthesis in cultured SVBalb/3T3 cells determined by the method of Hong and Levine (17,18). Triplicate cultures (2 x lo5 cells plated in 3.2 cm diameter dishes) were radiolabelled by incorporation from 1 pCi of [3H]-arachidonic acid during 24 hr in 2 ml DME containing 10% calf serum. About 60% of the added radioactivity is taken up by the cells and about 98% is converted to phospholipids and triglycerides. The cells were washed on the dishes 3 times with 2 ml DME, and the amount _+ SE of PGE2 (-0-) and total free fatty acid (-O-) radioactivity released in 30 min at 37O into 0.6 ml of DME containing the indicated concentrations of lysolecithin was determined by extracting and chromatographing lipids released into the medium using the method described in Fig. 1, and expressed as a percentage of the radioactivity incorporated into the cells. Under these conditions Q 92% of free arachidonic acid, 90% of PGE2 and 80% of PGF radioactivity was released into the culture medium. The amount of exogenous lysolecithin absorbed by the cells during 30 min at each concentration was estimated in parallel cultures by monitoring the percentage of [choline methyl-14C]-lysolecithin absorbed. The release f SE of PGE2 radioactivity during the next 30 min into an additional 0.6 ml DME containing bovine thrombin (10 pg/ml) (-El-) was determined in the same manner. Cell protein was 380 I-lg per dish.
cells
to PGE2 was observed
concentrations
b
5%.
tase enzyme system,
This
since
(see Fig. result
4) following is
treatment
not
with
treatment
due to saturation direct
lytic
with
calf
serum at
of the PG synthe-
factor
from African
Ringhals Cobra (30 pg/ml in DME) in parallel cultures stimulated the synthesis of 1.6 times as much PGE2 as the maximum stimulated by serum and thrombin (i.e.
at 5% serum). DISCUSSION A normal
be calculated This
amount
microsomes
basal from
lysolecithin-to-protein the
of lysolecithin (Fig.
data
ratio
of
Ray et al.
is
not
1) or in SVBalb/3T3
inhibitory cells
1172
(19)
equal for
rat
in either (Fig.
3).
to 0.0123 liver bovine
(w/w)
plasma
seminal
A lecithin-to-protein
can
membranes. vesicle
Vol. 78, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.Ii O-?i-
0.1
0.2
CONC.
OF
0.5
I
2
CALF
SERUM
5
IO
(%I
Fig. 4. Effect of stimulation of SVBalb/3T3 cells for 30 min by a range of -__ concentrations of calf serum on endogenous phospholipase activity3+ SE (-a-), and on the efficiency of conversion ? SE of the released [ H]arachidonic acid tc PGE2 during stimulation by serum alone (-U-) or in paralle cultures by serum with thrombin (10 pg/ml) (-0-), determined by the methods described in Fig. 3. The efficiency of conversion of free arachidonic acid to Serum-activated F-type PG exhibited similar changes with serum concentration. endogenous deacylating activity in SVBalb/3T3 cells generates endogenous lysolecithin as well as free fatty acids (22).
ratio
of 0.24
(w/w)
can be calculated
of Ray et al.
(19);
the lysolecithin-to-protein
inhibition
of PGE2 synthesis
Fig. 1B) would of the Lecithin Treatment labelled
cell
lipids
in about
1 hr
(5.8%
ments;
a similar
Il71). inhibits
Because
modify provide
acids
membranes that
vesicle
5% serum stimulates
that
would
30 min in Fig.
achieve
fatty
affect
acid
other
elevated
of
these
biochemical
levels enzymes basis
degree
enzymes, in membranes
in a coordinate for
(i.e.
a proposed-(l)
of prostaglandin synthesis by lysolecithin a possible explanation for the prostaglandin
by Manku tricyclic
and Horrobin
(20)
antidepressant
with
certain
local
and methylxanthine
1173
0.03,
of
of hydrolysis
2 hr in some experiof Hong and Levine
at which
activity
membrane-associated
lysolecithin
50%
hydrolysis of 12.5% ratio of 0.24 (w/w).
from the results
cycle-oxygenase
the data
the hydrolysis
this
ratios
causes
microsomes
4, and up to 25% in
can be calculated
from
lysolecithin
(Fig.
1) are
including would
similar
nucleotide be expected
fashion.
These
mechanism
regulation of membrane-associated enzymes by alteration lipid composition in response to external stimuli.
Inhibition offers
arrhythmic,
with
at a rate
value
additional
observed
seminal
the lysolecithin-to-protein
the activities
coordinate membrane
liver ratio
in bovine
cells
in
microsomal that (l),
rat
be produced by phospholipase A2 catalyzed in membranes with a lecithin-to-protein
of SVBalb/3T3
to those cyclases
for
and free antagonist anesthetic, compounds.
for
to
studies the
of cell fatty effects antiCompounds
Vol. 78, No. 4, 1977
of these
types
have been observed
A:lysolecithin elevated lower
acyltransferase levels
lysolecithin
BIOCHEMICAL
of lysolecithin may exhibit
PG levels
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be effective
activity
tissues
Acknowledgment - I gratefully Mr. J. T. Trotter.
from several
in treated
PG synthesis
inhibitors
tissues
without
and apparent
sources (21).
inhibiting
PG antagonist
acknowledge
the
of acyl
technical
coenzyme
and they
Elevated
cause
levels
degradation,
of
causing
effects. assistance
of
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Shier, W. T., Baldwin, J. H., Nilsen-Hamilton, M., Hamilton, R. and Thanassi, N. M. (1976) Proc. Natl. Acad. Sci. USA, 73, 1586-1590. Shier, W. T. and Trotter, J. T. (1976) Fed. Proc., 25, 1731. Shier, W. T. and Trotter, J. T. (1976) FEBS Lett., 62, 165-168. Kunze, H. and Vogt, W. (1971) Ann. N.Y. Acad. Sci., 180, 123-125. Samuelsson, B. (1972) Fed. Proc., 2, 1442-1450. Glass, D. B., Gerrard, J. M., Townsend, D., Carr, D. W., White, J. G. and Goldberg, N. D. (1977) .i. Cyclic Nucl. Res., 1, 37-44. Gorman, R. R., Hamberg, M. and Samuelsson, B. (1975) J. Biol. Chem., 25J, 6460-6463. Kuehl, F. A. (1974) Prostaglandins, 5, 325-340. Asakawa, T., Scheinbaum, I. and Ho, R. .I. (1976) Biochem. Biophys. Res. Commun., 12. 141-148. Hamberg, M., Svensson, J., Wakabayashi, T. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. USA, 71, 345-349. Robak, J., Dembinska-Kiec, A. and Gryglewski, R. (1975) Biochem. Pharm., 24, 2057-2060. Fryklund, L. and Eaker, D. (1973) Biochemistry, 12, 661-667. Wells, M. A. and Hanahan, D. J. (1969) Biochemistry, 8, 414-424. J. and Martin, D. B. (1972) J. Biol. Chem., --) 247 Carter, J. R., Avruch, 2682-2688. Flower, R. J., Cheung, H. S. and Cushman, D. W. (1973) Prostaglandins, 6, 325-341. Helenius, A. and Simons, K. (1975) Biochim. Biophys. Acta, 415, 29-79. Hong, S. L. and Levine, L. (1976) Proc. Natl. Acad. Sci. USA, 73, 1730-1734. Hong, S. L. and Levine, L. (1976) J. Biol. Chem., 251, 5814-5816. Ray, T. K., Skipski, V. P., Barclay, M., Essner, E. and Archibald, F.M. (1969) J. Biol. Chem., 2/&, 5528-5536. Manku, M. S. and Horrobin, D. F. (1976) Prostaglandins, 2, 789-801. Shier, W. T. (1977) Biochem. Biophys. Res. Commun., 2, 186-193. Shier, W. T., manuscript in preparation.
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