Vol. 179, No. 2, 1991 September 16, 1991
EFFECT
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 852-858
BIOCHEMICAL
OF OKADAIC ACID ON HORMONE- AND MASTOPARAN-STIMULATED TURNOVER IN ISOLATED RAT HEPATOCYTES
J. Adolf0
GarcTa-Sbinz,
Instituto
Received
Marina
de Fisiologfa
July
22,
MacIas-Silva
PHOSPHOINOSITIDE
and M. Teresa
Celular, UNAM, Apartado Me'xico D. F. 04510
Romero-Avila
Postal
70-248,
1991
Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angiotensin II and vasopressin. Mastoparan, a peptide toxin from wasp venom that mimics receptors by activating G-proteins, also the accumulation of stimulates inositol phosphates in hepatocytes. Interestingly,this action of mastoparan was also inhibited by okadaic acid. Our data indicate that okadaic acid inhibits the phosphoinositide turnover signal transduction system in hepatocytes at a level distal to the receptors. 0 1991 Academic Press, Inc.
Okadaic
acid,
dinoflagellates,
is
and 2A [1,2]. since
it
This seems
controlled
a
polyether
a potent compound
Protein through
which
molecular
is
cells
entities
for
is
regulate
in
of
regulation
signal
Using
isolated
C blocks
0006-291X/91 Copyright All rights
cyclase
[lo] rat
the
of G-proteins and phospholipase
hepatocytes
alphal-adrenergic
, we first action
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
852
[7-91
of
phosphatases
1
regulation
processes
that
common biochemical
[5].
61
types
are
[3,4].
The function
there and
is
mechanisms of some of the
seems to be
i.e., [see
phosphorylation
several
of cellular
biological
transduction
cycles,
by phosphorylation
as adenylate
kinase
receptor
study
of proteins
metabolism
by of protein
the
one of the most
phosphorylationjdephosphorylation evidence
for
identifying
their
involved
produced
inhibitor
a new probe
phosphorylation
phosphorylation
acid
and specific
to be useful
by reversible
fatty
modulated
a large
also
some
and membrane
by
amount
of
evidence
effecters,
of such
C [11,12]. showed
that
in hepatocytes
activation [13,14].
of protein The effect
Vol.
was
rather
signal or
BIOCHEMICAL
179, No. 2, 1991
selective
tranduction affected
since
the effects
system
that
the
kinase
C activation Here,
blockade is
we
turnover
signal
indicate
that
extent
associated the
agent
coupled
turnover)
were
Using
smooth
[13,14].
action
to alphal-adrenoceptor
effect
transduction
RESEARCH COMMUNICATIONS
receptors
of the alphal-adrenergic
report
this
of other
( phosphoinositide
to a much lesser
shown
AND BIOPHYSICAL
of okadaic
system alters
in
the
on
rat
function
not
affected
muscle
cells
it
induced
by
the
system
was
protein [18].
phosphoinositide
hepatocytes.
of the
same
either
phosphorylation
acid
isolated
to the
Our
at
a
results
postreceptor
site. MATERIALS
AND METHODS
1-Epinephrine, angiotensin II, vasopressin and mastoparan were obtained from Sigma Chemical Co. 2-13H] -My0 inositol (20 Ci/mmol) and [32P]Pi (carrier free) were obtained from New England Nuclear. The anion exchange resin AG l-X8 (formate form) was from BioRad and collagenase from Worthington. Okadaic acid was from Gibco (the initial experiments were performed with a sample of this tumor promoter generously provided by Dr. Philip Cohen, (Dundee, U.K.)). libitum. cells buffer, shaker.
Experiments were performed with female Wistar rats (220-250 g) fed ad Hepatocytes were isolated by the method of Berry and Friend [19]. The (30-40 mg wet weight) were incubated in 1 ml of Krebs-Ringer bicarbonate under an atmosphere of 95% 021 5% C02, pH 7.4 at 37' C in a water bath
Phosphatidylinositol labeling was studied as described previously [20]. Production of inositol phosphates was studied as described [21,22]. In brief, the cells were incubated for 90 min with 15 nCi/ml of tritiated inositol, washed and incubated for 10 min with 10 mM LiCl in the presence or absence of okadaic acid; the hormones were added and after 5 min of incubation the reaction was stopped. Inositol phosphates were separated by anion exchange chromatography as described by Berridge et al counts in [231. The total fractions l-4 (IPl),6-8 (IP2) and 11-12 (IP3) were added to determine the production of these inositol phosphates. RESULTS In
cells
increase Okadaic
in
incubated the
acid
in basal
labeling).
To
promoter,
we took
as percentage parallel
labeling
by itself
+ 3% decrease
with
with
study into
of their the
[32P]Pi,
epinephrine
of phosphatidylinositol decreases labeling the
the
labeling
at 1 nM okadaic
effect
account
basal
(PI)
of hormones this
own basal,
same concentration
decrease i.e.
the
853
a
dose-dependent
(Fig.
1,
left
panel).
of phosphatidylinositol acid;
in
cells
in
labeling
labeling
of okadaic
induced
acid.
n=7, incubated
(33
p < 0.005 with
this
and expressed observed Under
in these
vs basal tumor
the
cells
run
conditions,
data in
Vol.
179. No.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-LOG [EPINEPHRINE]M
-LOG [OKADAIC ACID] M Figure 1. EFFECT OF OKADAIC ACID ON HORMONAL STIMULATION OF PHOSPHATIDYL INOSITOL (PI) LABELING. Left Panel: cells were incubated in the absence or presence (O---O) of 1 u M Okadaic c-1 Acid and with different concentrations of epinephrine plus 10 PM propranolol. Middle panel: cells were incubated with 10 nM epinephrine plus 10 PM propranolol alone (0) or with (0) different concentrations of okadaic acid. Right panel: cells were incubated without (solid bars) or with (open bars) 1 PM Okadaic Acid and the agonists: EPI, 10 UM epinephrine plus 10 !JM propranolol; VASO, 10 nM vasopressin; ANGIO, 10 PM angiotensin II. Results are the means and vertical lines represent the S.E.M. of 4-5 experiments in duplicate. we
observed
the
alphal-adrenergic
middle
that
okadaic effect
panel);
adrenergic left
the
agonist
panel).
effect, PI
inhibited
and
labeling, to a similar
We confirmed
this
phosphates.
Okadaic
of
phosphates.
of epinephrine
(IC50
fatty
Interestingly,
acid
affecting action
the
such
as
vasopressin
extent
acid
was without
the
effect it
effect
acid
(Fig.
(Fig.
3 and Table
1
of
the
(Fig.
1,
for
the
that
II,
was
also also
panel).
the production
markedly
of these
selective
hormones
1, right
by itself
induced
(Fig.
effect
angiotensin
or
and angiotensin
of IP2 and IP3 production
by okadaic
X)
was not
of other
by quantifying
In contrast,
only
action
( 40-60
vasopressin
not
acid
of
acid)
( =3 pM epinephrine)
of okadaic
e.
inhibition
the maximal
EC50
i.
partial
z 50 nM okadaic
altered
its
observation
by epinephrine,
stimulation
a dose-dependent
The inhibitory
stimulate
induced
produced
polyether without
alphal-adrenergic
inositol
acid
on the
of basal
attenuated II
(Fig.
hormones
was
by mastoparan
the
inositol production
stimulations
2 and Table altered was
also
but
1). the
inhibited
2).
DISCUSSION Our
present
phosphoinositide
results turnover
indicate signal
that
okadaic
transduction 854
acid system
markedly in
alters
hepatocytes.
the Two
Vol.
179,
No.
BIOCHEMICAL
2, 1991
AND
VASO IP,
I
IP,
I
I
IP3
IP,
RIOPHYSICAL
ANQIO I IP, ’ IP,
FRACTION
RESEARCH
COMMUNICATIONS
EPI IP,
’ IP,
’ LFy
NUMBER
Figure 2. ELUTION PROFILE OF INOSITOL PHOSPHATES. Cells were incubated in the absence of any agent (M) with 1 I,IM Okadaic Acid (O---O) with the agonists (VASO, 10 nM vasopressin; ANGIO, 10 !J M angiotensin II or EPI, 10 UM epinephrine plus 10 u M propranolol) (M) or with the agonists plus 1 u M okadaic acid (A---A). Note left ordinate for EPI-IP3. Data are from a representative experiment in triplicate.
EFFECTS
OF OKADAIC
TABLE 1 ACID, EPINEPHRINE, VASOPRESSIN AND ANGIOTENSIN [3H]INOSITOL PHOSPHATES ACCUMULATION
Condition
Inositol
Phosphates
(cpm/40
IPl Basal
mg cells
IP2
IP3
-+ 160
400
1 PM
2320
2 210
385 -I- 35
135 + 10
Epinephrine
10 DM
2690
-+ 175
740
+ 90a
210
-+
zob
Epinephrine
10 UM + 2340
+ 215
510
t
6OC
150
2
lad
3085
k 125a
+ z3ob
610
+-
95b
1 UM
2590
-+ 17oc
990 + 135c
320
+
49
Vasopressin
10 nM
3065
5 130a
660
t
85b
Vasopressin
10 nM + 2345
2 155e
325
+
50e
Acid
Okadaic
Acid
1uM
Angiotensin
II
10 UM
Angiotensin
II
10 PM +
Okadaic
Acid
Okadaic are
ap < 0.005 vs hormone
Acid the
means
1 !.IM 2 S.E.M.
of
8 experiments
1650
1635
+- 40
+ 16ob
740 +- 60f in
125
+-
5
triplicate.
vs basal; bp < 0.001 vs basal; cp < 0.05 vs hormone alone; alone; ep < 0.005 vs hormone alone; fp < 0.001 vs hormone
855
ON
w.w.)
2300
Okadaic
Results
II
dp < 0.02 alone.
Vol.
179, No. 2, 1991
BiOCHEMlCAL
AND BIOPHYSICAL
1000
RESEARCH COMMUNICATIONS
400 350
800
300 5a
600
0 400
250
I
200
El
150
200
100 50 5 FRACTION
IO I5 NUMBER
Figure 3. ELUTION PROFILE OF INOSITOL PHOSPHATES. absence of any agent (U) with 1 nM Okadaic mastoparan (s) or with 50 nM mastoparan plus Note left ordinate for IP2 and IP3. Data are from in triplicate.
different, finding. (PI
but
complementary
i.e., labeling)
hydrolysis of
entirely
consistent
partially
inhibits
The acetate
(production
and indicate this of Using
signal okadaic
were
The that
this
transduction acid
hepatocytes,
determined
of inositol
phosphoinositides.
effect (PMA).
parameters
Cells were incubated in the Acid (O---O) with 50 n M 1 PM Okadaic acid (A---A). a representative experiment
document
phosphates)
results
of
inhibitor
and resynthesis
both
of
to that
has been
observed
TABLE 2 EFFECTS OF ORADAIC ACID AND MASTOPARAN ON [3H]IP3 Condition
parameters
protein
of
phorbol that
Basal
ACCDMULATION
50 nM
Mastoparan
50 pM +
Okadaic
315 -+ 20
125 -+ 5
555 -+ 25a
175 -+ 10a
440 -+ 2+
155 2 10
1nM
Mastoparan
Acid 1 pM
Results are the means 2 S.E.M. of 7 experiments a p < 0.001 vs basal: b p < 0.01 vs mastoparan 856
in triplicate. alone.
myristate
activation
Inositol Phosphates (cpm / 40 mg cells w.w.)
Acid
were
phosphatases
IP3
Okadaic
this
process.
was different it
to
of
BIOCHEMICAL
Vol. 179, No. 2, 1991
protein
kinase
action
[13-171
contrast,
C by PMA leads with
the
receptor
only
selectivity
to a complete
minor
inhibition
AND BIOPHYSICAL
effects
induced was observed.
okadaic
acid
is
(are)
not
entities
involved
protein
or phospholipase
exerted
in steps
blockade
on that
of the
of other
by okadaic
acid
These
suggest
data
at the
subsequent C.
RESEARCH COMMUNICATIONS
level
with
hormones
[13-171.
was rather
broad
that
the
of the hormone
to receptor
The data
alphal-adrenergic
mastoparan
and
of but
possibly
are
no
action(s)
receptors
activation,
In
on
the
consistent
with
Gthis
interpretation. Mastoparan stimulator
promoting used
by
receptors of
accumulation.
We
mastoparan acid
of
okadaic
acid level
take
the
G-protein,
C. results
in
phosphoinositide
C- B by
phosphorylation It
1121. this
study
controlling
our
transduction
can
steady
Experiments where
data
we
the
indicate
C may alter
system
however,
not
rule
state
levels
of
in progress,
alteration(s)
induced okadaic
in hepatocytes
changes
alters
at a level 857
that
the
system
induced
by
but
rather
at
acts, of
been with
the
cell-free
the distal
on
cells
the
and of
take
of and
that
G-protein used enzymes inositol
to define
place.
phosphoinositide to the
of
approach
activity
systems,
acid
or
suggested
phospholipids
by okadaic
acid
suggest
the whole-cell
inositol using
has
in
pM
of phosphorylation
interaction
with
50
stimulation
evidence
its
IP3
IP3.
putatively
and it
that
out
are
that
C [12]
and
IP2 and
C in a variety
is
high
that
of
receptor-mediated
There
kinase
be mentioned,
the
phosphates. site(s)
protein
of
observed
production
to
that
calcium
further
on G-
similar
of the receptors
kinase
301.
free
mastoparan
potent
seems to act
transduction
level
protein
29,
of phospholipase should
at the on which
[12,
data
turnover
inhibition
turnover
phospholipase
These
a
observed,
and the
is
strikingly
[28]
finding
effect.
of
the
al
consistently
place
Activation
Mastoparan
cytosolic
later
but
this
et
increase
the phosphoinositide
of
tissues
modestly
wasp venom that
by a mechanism
Tohkin
this
does not
phospholipase
in
confirmed
from
[24-261.
exchange
[27].
inhibited
perturbation
cells
mastoparan
increases
Okadaic
toxin
in several nucleotide
concentrations,
the
a tetradecapeptide
of secretion
proteins that
is
receptors.
the
In summary, turnover
Vol.
179,
No.
BIOCHEMJCAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS The authors They also was partially
thank
thank Ms. supported
Dr. Guadalupe
Philip
Cohen
Ramirez
by a Grant
for
for
from
his typing
DGAPA (IN
generous
gift
the manuscript.
of okadaic This
acid. research
201889).
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Bialojan, C., and Takai, A. (1988) Biochem. J. 256, 283-290. Heschler, J. Mieskes, G., Ruegg, J.C., Takai, A. and Trautwein, W. (1988) Pflugers Arch. Ges. Physiol. 412, 248-252. Haystead, T.A.J., Sim, A.T.R., Carling, D., Honnor, R.C, Tsukitani, Y., Cohen, P. and Hardie, D.G. (1989) Nature 337, 78-81. Cohen, P., Holmes, C.F.B. and Tsukitani, Y. (1990) Trends Biochem. Sci. 15, 98-102. Cohen, P. (1982) Nature 296, 613-620 Moudgil, V.K. (1989) Receptor Phosphorylation. CRC Press, Inc. U.S.A. Katada, T., Gilman, A.G., Watanabe, Y., Bauer, S. and Jakobs, K.H. (1985) Eur. J. Biochem. 151, 431-437. Houslay, M.D. (1991) Cell. Signal. 3, l-9. Bushfield, M., Lavan, B.E. and Houslay, M.D. (1991) Biochem. J. 274, 317321. Yoshimasa, T., Sibley, D.R., Bouvier, M., Lefkowitz, R.J. and Caron, M.G. (1987) Nature 327, 67-70. Nishibe, S., Wahl, M.I., Hernsndez-Sotomayor,S.M.T., Tonks, N.K., Rhee, S.G., Carpenter, G. (1990) Science 250, 1253-1256. Ryu, S.H., Kim, U-H., Wahl, M.I., Brown, A.B., Carpenter, G., Huang, K-P., and Rhee, S.G. (1990) J. Biol. Chem. 265, 17941-17945. Corvera, S. and Garcia-Sa'inz, J.A. (1984) Biochem. Biophys. Res. Commun. 119, 1128-1133. Corvera, S., Schwarz, K.R., Graham, R.M. and Garcfa-Sginz, J.A. (1986) J. Biol. Chem. 261, 520-526. Lynch, C.J., Charest, R., Bocckino, S-B.,' Exton, J.H. and Blackmore, P.F. (1985) J. Biol. Chem. 260, 2844-2851. Cooper, R.H., Coll, K.E. and Williamson, J.R. (1985) J. Biol. Chem. 260, 3281-3288. Van de Werve, G., Proietto, J. and Jeanrenaud, B. (1985) Biochem. J. 231, 511-516. Leeb-Lundberg, L.M.F., Cotecchia, S., Lomasney, J.W., DeBernardis, J.F., Lefkowitz, R.J. and Caron, M.G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655. Berry, M.N. and Friend, D.S. (1969) The Journal of Cell Biology 43, 506520. Garcia-SBinz, J.A. (1987) Circ. Res. 61 (Suppl. II) 1-5. Charest, R., Prpic, V., Exton, J.H. and Blackmore, P.F. (1985) Biochem. J. 227, 79-90. Garcia-Szinz, J.A. and Macias-Silva, M. (1990) Biochem. Biophys. Res. Commun. 172, 780-785. Dawson, R.M., Downes, P., Heslop, J.P. and Irvine, R.F. Berridge, M.J., (1983) Biochem. J. 212, 473-482. Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M., and Hirai, Y., Kitada, C. (1979) Chem. Pharm. Bull. (Tokyo) 27, 1942-1944. Kuroda, Y., Yoshioka, M., Kumajura, K., Kobayashi, K. and Nakajima, T. (1980) Proc. Jpn. Acad. Ser. B 56, 660-664. Kurihara, H., Kitajima, K.. Senda, T., Fujita, H. and Nakajima, T. (1986) Cell. Tissue Res. 243, 311-316. Higashijima, T., uzu, s., Nakajima, T., and Ross, E.M. (1988) J. Biol. Chem. 263, 6491-6494. Tohkin, M., Yagami, T., and Matsubara, T. (1990) FEBS Lett 260, 179-182. Blackmore, P.F., and Exton, J.H. (1986) J. Biol. Chem. 261, 11056-11063. Orellana, S., Solski, P.A., and Brown, J.H. (1987) J. Biol. C&m. 262, 1638-1643.
858
EFFECT
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 852-858
BIOCHEMICAL
OF OKADAIC ACID ON HORMONE- AND MASTOPARAN-STIMULATED TURNOVER IN ISOLATED RAT HEPATOCYTES
J. Adolf0
GarcTa-Sbinz,
Instituto
Received
Marina
de Fisiologfa
July
22,
MacIas-Silva
PHOSPHOINOSITIDE
and M. Teresa
Celular, UNAM, Apartado Me'xico D. F. 04510
Romero-Avila
Postal
70-248,
1991
Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angiotensin II and vasopressin. Mastoparan, a peptide toxin from wasp venom that mimics receptors by activating G-proteins, also the accumulation of stimulates inositol phosphates in hepatocytes. Interestingly,this action of mastoparan was also inhibited by okadaic acid. Our data indicate that okadaic acid inhibits the phosphoinositide turnover signal transduction system in hepatocytes at a level distal to the receptors. 0 1991 Academic Press, Inc.
Okadaic
acid,
dinoflagellates,
is
and 2A [1,2]. since
it
This seems
controlled
a
polyether
a potent compound
Protein through
which
molecular
is
cells
entities
for
is
regulate
in
of
regulation
signal
Using
isolated
C blocks
0006-291X/91 Copyright All rights
cyclase
[lo] rat
the
of G-proteins and phospholipase
hepatocytes
alphal-adrenergic
, we first action
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
852
[7-91
of
phosphatases
1
regulation
processes
that
common biochemical
[5].
61
types
are
[3,4].
The function
there and
is
mechanisms of some of the
seems to be
i.e., [see
phosphorylation
several
of cellular
biological
transduction
cycles,
by phosphorylation
as adenylate
kinase
receptor
study
of proteins
metabolism
by of protein
the
one of the most
phosphorylationjdephosphorylation evidence
for
identifying
their
involved
produced
inhibitor
a new probe
phosphorylation
phosphorylation
acid
and specific
to be useful
by reversible
fatty
modulated
a large
also
some
and membrane
by
amount
of
evidence
effecters,
of such
C [11,12]. showed
that
in hepatocytes
activation [13,14].
of protein The effect
Vol.
was
rather
signal or
BIOCHEMICAL
179, No. 2, 1991
selective
tranduction affected
since
the effects
system
that
the
kinase
C activation Here,
blockade is
we
turnover
signal
indicate
that
extent
associated the
agent
coupled
turnover)
were
Using
smooth
[13,14].
action
to alphal-adrenoceptor
effect
transduction
RESEARCH COMMUNICATIONS
receptors
of the alphal-adrenergic
report
this
of other
( phosphoinositide
to a much lesser
shown
AND BIOPHYSICAL
of okadaic
system alters
in
the
on
rat
function
not
affected
muscle
cells
it
induced
by
the
system
was
protein [18].
phosphoinositide
hepatocytes.
of the
same
either
phosphorylation
acid
isolated
to the
Our
at
a
results
postreceptor
site. MATERIALS
AND METHODS
1-Epinephrine, angiotensin II, vasopressin and mastoparan were obtained from Sigma Chemical Co. 2-13H] -My0 inositol (20 Ci/mmol) and [32P]Pi (carrier free) were obtained from New England Nuclear. The anion exchange resin AG l-X8 (formate form) was from BioRad and collagenase from Worthington. Okadaic acid was from Gibco (the initial experiments were performed with a sample of this tumor promoter generously provided by Dr. Philip Cohen, (Dundee, U.K.)). libitum. cells buffer, shaker.
Experiments were performed with female Wistar rats (220-250 g) fed ad Hepatocytes were isolated by the method of Berry and Friend [19]. The (30-40 mg wet weight) were incubated in 1 ml of Krebs-Ringer bicarbonate under an atmosphere of 95% 021 5% C02, pH 7.4 at 37' C in a water bath
Phosphatidylinositol labeling was studied as described previously [20]. Production of inositol phosphates was studied as described [21,22]. In brief, the cells were incubated for 90 min with 15 nCi/ml of tritiated inositol, washed and incubated for 10 min with 10 mM LiCl in the presence or absence of okadaic acid; the hormones were added and after 5 min of incubation the reaction was stopped. Inositol phosphates were separated by anion exchange chromatography as described by Berridge et al counts in [231. The total fractions l-4 (IPl),6-8 (IP2) and 11-12 (IP3) were added to determine the production of these inositol phosphates. RESULTS In
cells
increase Okadaic
in
incubated the
acid
in basal
labeling).
To
promoter,
we took
as percentage parallel
labeling
by itself
+ 3% decrease
with
with
study into
of their the
[32P]Pi,
epinephrine
of phosphatidylinositol decreases labeling the
the
labeling
at 1 nM okadaic
effect
account
basal
(PI)
of hormones this
own basal,
same concentration
decrease i.e.
the
853
a
dose-dependent
(Fig.
1,
left
panel).
of phosphatidylinositol acid;
in
cells
in
labeling
labeling
of okadaic
induced
acid.
n=7, incubated
(33
p < 0.005 with
this
and expressed observed Under
in these
vs basal tumor
the
cells
run
conditions,
data in
Vol.
179. No.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-LOG [EPINEPHRINE]M
-LOG [OKADAIC ACID] M Figure 1. EFFECT OF OKADAIC ACID ON HORMONAL STIMULATION OF PHOSPHATIDYL INOSITOL (PI) LABELING. Left Panel: cells were incubated in the absence or presence (O---O) of 1 u M Okadaic c-1 Acid and with different concentrations of epinephrine plus 10 PM propranolol. Middle panel: cells were incubated with 10 nM epinephrine plus 10 PM propranolol alone (0) or with (0) different concentrations of okadaic acid. Right panel: cells were incubated without (solid bars) or with (open bars) 1 PM Okadaic Acid and the agonists: EPI, 10 UM epinephrine plus 10 !JM propranolol; VASO, 10 nM vasopressin; ANGIO, 10 PM angiotensin II. Results are the means and vertical lines represent the S.E.M. of 4-5 experiments in duplicate. we
observed
the
alphal-adrenergic
middle
that
okadaic effect
panel);
adrenergic left
the
agonist
panel).
effect, PI
inhibited
and
labeling, to a similar
We confirmed
this
phosphates.
Okadaic
of
phosphates.
of epinephrine
(IC50
fatty
Interestingly,
acid
affecting action
the
such
as
vasopressin
extent
acid
was without
the
effect it
effect
acid
(Fig.
(Fig.
3 and Table
1
of
the
(Fig.
1,
for
the
that
II,
was
also also
panel).
the production
markedly
of these
selective
hormones
1, right
by itself
induced
(Fig.
effect
angiotensin
or
and angiotensin
of IP2 and IP3 production
by okadaic
X)
was not
of other
by quantifying
In contrast,
only
action
( 40-60
vasopressin
not
acid
of
acid)
( =3 pM epinephrine)
of okadaic
e.
inhibition
the maximal
EC50
i.
partial
z 50 nM okadaic
altered
its
observation
by epinephrine,
stimulation
a dose-dependent
The inhibitory
stimulate
induced
produced
polyether without
alphal-adrenergic
inositol
acid
on the
of basal
attenuated II
(Fig.
hormones
was
by mastoparan
the
inositol production
stimulations
2 and Table altered was
also
but
1). the
inhibited
2).
DISCUSSION Our
present
phosphoinositide
results turnover
indicate signal
that
okadaic
transduction 854
acid system
markedly in
alters
hepatocytes.
the Two
Vol.
179,
No.
BIOCHEMICAL
2, 1991
AND
VASO IP,
I
IP,
I
I
IP3
IP,
RIOPHYSICAL
ANQIO I IP, ’ IP,
FRACTION
RESEARCH
COMMUNICATIONS
EPI IP,
’ IP,
’ LFy
NUMBER
Figure 2. ELUTION PROFILE OF INOSITOL PHOSPHATES. Cells were incubated in the absence of any agent (M) with 1 I,IM Okadaic Acid (O---O) with the agonists (VASO, 10 nM vasopressin; ANGIO, 10 !J M angiotensin II or EPI, 10 UM epinephrine plus 10 u M propranolol) (M) or with the agonists plus 1 u M okadaic acid (A---A). Note left ordinate for EPI-IP3. Data are from a representative experiment in triplicate.
EFFECTS
OF OKADAIC
TABLE 1 ACID, EPINEPHRINE, VASOPRESSIN AND ANGIOTENSIN [3H]INOSITOL PHOSPHATES ACCUMULATION
Condition
Inositol
Phosphates
(cpm/40
IPl Basal
mg cells
IP2
IP3
-+ 160
400
1 PM
2320
2 210
385 -I- 35
135 + 10
Epinephrine
10 DM
2690
-+ 175
740
+ 90a
210
-+
zob
Epinephrine
10 UM + 2340
+ 215
510
t
6OC
150
2
lad
3085
k 125a
+ z3ob
610
+-
95b
1 UM
2590
-+ 17oc
990 + 135c
320
+
49
Vasopressin
10 nM
3065
5 130a
660
t
85b
Vasopressin
10 nM + 2345
2 155e
325
+
50e
Acid
Okadaic
Acid
1uM
Angiotensin
II
10 UM
Angiotensin
II
10 PM +
Okadaic
Acid
Okadaic are
ap < 0.005 vs hormone
Acid the
means
1 !.IM 2 S.E.M.
of
8 experiments
1650
1635
+- 40
+ 16ob
740 +- 60f in
125
+-
5
triplicate.
vs basal; bp < 0.001 vs basal; cp < 0.05 vs hormone alone; alone; ep < 0.005 vs hormone alone; fp < 0.001 vs hormone
855
ON
w.w.)
2300
Okadaic
Results
II
dp < 0.02 alone.
Vol.
179, No. 2, 1991
BiOCHEMlCAL
AND BIOPHYSICAL
1000
RESEARCH COMMUNICATIONS
400 350
800
300 5a
600
0 400
250
I
200
El
150
200
100 50 5 FRACTION
IO I5 NUMBER
Figure 3. ELUTION PROFILE OF INOSITOL PHOSPHATES. absence of any agent (U) with 1 nM Okadaic mastoparan (s) or with 50 nM mastoparan plus Note left ordinate for IP2 and IP3. Data are from in triplicate.
different, finding. (PI
but
complementary
i.e., labeling)
hydrolysis of
entirely
consistent
partially
inhibits
The acetate
(production
and indicate this of Using
signal okadaic
were
The that
this
transduction acid
hepatocytes,
determined
of inositol
phosphoinositides.
effect (PMA).
parameters
Cells were incubated in the Acid (O---O) with 50 n M 1 PM Okadaic acid (A---A). a representative experiment
document
phosphates)
results
of
inhibitor
and resynthesis
both
of
to that
has been
observed
TABLE 2 EFFECTS OF ORADAIC ACID AND MASTOPARAN ON [3H]IP3 Condition
parameters
protein
of
phorbol that
Basal
ACCDMULATION
50 nM
Mastoparan
50 pM +
Okadaic
315 -+ 20
125 -+ 5
555 -+ 25a
175 -+ 10a
440 -+ 2+
155 2 10
1nM
Mastoparan
Acid 1 pM
Results are the means 2 S.E.M. of 7 experiments a p < 0.001 vs basal: b p < 0.01 vs mastoparan 856
in triplicate. alone.
myristate
activation
Inositol Phosphates (cpm / 40 mg cells w.w.)
Acid
were
phosphatases
IP3
Okadaic
this
process.
was different it
to
of
BIOCHEMICAL
Vol. 179, No. 2, 1991
protein
kinase
action
[13-171
contrast,
C by PMA leads with
the
receptor
only
selectivity
to a complete
minor
inhibition
AND BIOPHYSICAL
effects
induced was observed.
okadaic
acid
is
(are)
not
entities
involved
protein
or phospholipase
exerted
in steps
blockade
on that
of the
of other
by okadaic
acid
These
suggest
data
at the
subsequent C.
RESEARCH COMMUNICATIONS
level
with
hormones
[13-171.
was rather
broad
that
the
of the hormone
to receptor
The data
alphal-adrenergic
mastoparan
and
of but
possibly
are
no
action(s)
receptors
activation,
In
on
the
consistent
with
Gthis
interpretation. Mastoparan stimulator
promoting used
by
receptors of
accumulation.
We
mastoparan acid
of
okadaic
acid level
take
the
G-protein,
C. results
in
phosphoinositide
C- B by
phosphorylation It
1121. this
study
controlling
our
transduction
can
steady
Experiments where
data
we
the
indicate
C may alter
system
however,
not
rule
state
levels
of
in progress,
alteration(s)
induced okadaic
in hepatocytes
changes
alters
at a level 857
that
the
system
induced
by
but
rather
at
acts, of
been with
the
cell-free
the distal
on
cells
the
and of
take
of and
that
G-protein used enzymes inositol
to define
place.
phosphoinositide to the
of
approach
activity
systems,
acid
or
suggested
phospholipids
by okadaic
acid
suggest
the whole-cell
inositol using
has
in
pM
of phosphorylation
interaction
with
50
stimulation
evidence
its
IP3
IP3.
putatively
and it
that
out
are
that
C [12]
and
IP2 and
C in a variety
is
high
that
of
receptor-mediated
There
kinase
be mentioned,
the
phosphates. site(s)
protein
of
observed
production
to
that
calcium
further
on G-
similar
of the receptors
kinase
301.
free
mastoparan
potent
seems to act
transduction
level
protein
29,
of phospholipase should
at the on which
[12,
data
turnover
inhibition
turnover
phospholipase
These
a
observed,
and the
is
strikingly
[28]
finding
effect.
of
the
al
consistently
place
Activation
Mastoparan
cytosolic
later
but
this
et
increase
the phosphoinositide
of
tissues
modestly
wasp venom that
by a mechanism
Tohkin
this
does not
phospholipase
in
confirmed
from
[24-261.
exchange
[27].
inhibited
perturbation
cells
mastoparan
increases
Okadaic
toxin
in several nucleotide
concentrations,
the
a tetradecapeptide
of secretion
proteins that
is
receptors.
the
In summary, turnover
Vol.
179,
No.
BIOCHEMJCAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS The authors They also was partially
thank
thank Ms. supported
Dr. Guadalupe
Philip
Cohen
Ramirez
by a Grant
for
for
from
his typing
DGAPA (IN
generous
gift
the manuscript.
of okadaic This
acid. research
201889).
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Bialojan, C., and Takai, A. (1988) Biochem. J. 256, 283-290. Heschler, J. Mieskes, G., Ruegg, J.C., Takai, A. and Trautwein, W. (1988) Pflugers Arch. Ges. Physiol. 412, 248-252. Haystead, T.A.J., Sim, A.T.R., Carling, D., Honnor, R.C, Tsukitani, Y., Cohen, P. and Hardie, D.G. (1989) Nature 337, 78-81. Cohen, P., Holmes, C.F.B. and Tsukitani, Y. (1990) Trends Biochem. Sci. 15, 98-102. Cohen, P. (1982) Nature 296, 613-620 Moudgil, V.K. (1989) Receptor Phosphorylation. CRC Press, Inc. U.S.A. Katada, T., Gilman, A.G., Watanabe, Y., Bauer, S. and Jakobs, K.H. (1985) Eur. J. Biochem. 151, 431-437. Houslay, M.D. (1991) Cell. Signal. 3, l-9. Bushfield, M., Lavan, B.E. and Houslay, M.D. (1991) Biochem. J. 274, 317321. Yoshimasa, T., Sibley, D.R., Bouvier, M., Lefkowitz, R.J. and Caron, M.G. (1987) Nature 327, 67-70. Nishibe, S., Wahl, M.I., Hernsndez-Sotomayor,S.M.T., Tonks, N.K., Rhee, S.G., Carpenter, G. (1990) Science 250, 1253-1256. Ryu, S.H., Kim, U-H., Wahl, M.I., Brown, A.B., Carpenter, G., Huang, K-P., and Rhee, S.G. (1990) J. Biol. Chem. 265, 17941-17945. Corvera, S. and Garcia-Sa'inz, J.A. (1984) Biochem. Biophys. Res. Commun. 119, 1128-1133. Corvera, S., Schwarz, K.R., Graham, R.M. and Garcfa-Sginz, J.A. (1986) J. Biol. Chem. 261, 520-526. Lynch, C.J., Charest, R., Bocckino, S-B.,' Exton, J.H. and Blackmore, P.F. (1985) J. Biol. Chem. 260, 2844-2851. Cooper, R.H., Coll, K.E. and Williamson, J.R. (1985) J. Biol. Chem. 260, 3281-3288. Van de Werve, G., Proietto, J. and Jeanrenaud, B. (1985) Biochem. J. 231, 511-516. Leeb-Lundberg, L.M.F., Cotecchia, S., Lomasney, J.W., DeBernardis, J.F., Lefkowitz, R.J. and Caron, M.G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655. Berry, M.N. and Friend, D.S. (1969) The Journal of Cell Biology 43, 506520. Garcia-SBinz, J.A. (1987) Circ. Res. 61 (Suppl. II) 1-5. Charest, R., Prpic, V., Exton, J.H. and Blackmore, P.F. (1985) Biochem. J. 227, 79-90. Garcia-Szinz, J.A. and Macias-Silva, M. (1990) Biochem. Biophys. Res. Commun. 172, 780-785. Dawson, R.M., Downes, P., Heslop, J.P. and Irvine, R.F. Berridge, M.J., (1983) Biochem. J. 212, 473-482. Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M., and Hirai, Y., Kitada, C. (1979) Chem. Pharm. Bull. (Tokyo) 27, 1942-1944. Kuroda, Y., Yoshioka, M., Kumajura, K., Kobayashi, K. and Nakajima, T. (1980) Proc. Jpn. Acad. Ser. B 56, 660-664. Kurihara, H., Kitajima, K.. Senda, T., Fujita, H. and Nakajima, T. (1986) Cell. Tissue Res. 243, 311-316. Higashijima, T., uzu, s., Nakajima, T., and Ross, E.M. (1988) J. Biol. Chem. 263, 6491-6494. Tohkin, M., Yagami, T., and Matsubara, T. (1990) FEBS Lett 260, 179-182. Blackmore, P.F., and Exton, J.H. (1986) J. Biol. Chem. 261, 11056-11063. Orellana, S., Solski, P.A., and Brown, J.H. (1987) J. Biol. C&m. 262, 1638-1643.
858
