Vol. 182, No. 2, 1992 January 31, 1992
ISOFLAVONOIDS,
Takeshi
Tomonaga', Tetsuya Mine', Haruyuki Hayashi'**, of
'Surgery Medicine,
Department of Medicine,
31nstitute
Received
AND BIOPHYSICAL
GENISTEIN, PSI-TECTORIGENIN, FREE CALCIUM IN ISOLATED
Departments 'Fourth
BIOCHEMICAL
RESEARCH COMMUNICATIONS Pages 894-899
AND OROBOL, INCREASE CYTOPLASMIC RAT HEPATOCYTES
Itaru Kojima3, Masanori and Kaichi Isono'
and 4Biochemistry, Chiba University 1-8-1 Inohana, Chiba 280, Japan
School
of
of Internal Medicine, University of Tokyo School 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan
of Endocrinology, Gunma University, 3-39-15 Maebashi, Gunma 371, Japan
December
Taira4*,
10,
Showa-machi,
1991
psi-tectorigenin and orobol have SUMMARY: Isoflavonoid compounds, genistein, been implicated as inhibitors of tyrosine-specific protein kinase and phosphatidylinositol turnover. These compounds have been frequently used as a pharmacological tool to assess signal transduction pathways in various cell systems. In the course of analyzing signaling pathways in rat hepatocytes, we obtained an unexpected finding that these corn ounds transiently increase cytoplasmic free calcium. Since the CaZP mobilizing effect was observed in 1 uM calcium containing buffer, the source of the Ca2+ may be intracellular stores. Thus, when interpreting data obtained 0 1992 Academic Press, Inc. using these compounds, caution is needed.
Multi-step factor
signaling
activation
Inhibitors molecular
of
of each
inhibitor
in
of isoflavonoids,
including (PI)
involved receptor
signaling
in
compound,
tyrosine-specific
are
surface
involved
of
phosphatidylinositol
cell
component
mechanisms
An isoflavonoid
the
events
the
protein
transduction
leading
to
of
nuclear
should
aid
growth
events.
in elucidating
transduction. was first
kinase
psi-tectorigenin turnover
the
pathways
signal
genistein
in
was also
(1).
designated The inhibitory
and orobol, reported
as an
(2-6).
effects
on Other
workers
*Present address: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. *qo whom correspondence should be addressed. Abbreviations:PI, phosphatidylinositol; EGTA, Ethylene Glycol Bis-(Baminoethyl ether) N,N,N',N'-tetraacetic Acid. ooo6-291x/92 Copyright All rights
$1.50
0 1992 by Academic Press, of reproduction in any form
Inc. resewed.
894
Vol.
182, No. 2, 1992
noted
the
These
compounds
pathways
BIOCHEMICAL
inhibition
even
of have
though
AND BIOPHYSICAL
topoisomerase
II
been frequently specificity
(7-9)
used of
the
or 56 kinase
to assess
action
RESEARCH COMMUNICATIONS
of
signal these
(10)
activity.
transduction compounds
is
not
clear. In the expression
course
of analyzing
signaling
and DNA synthesis
transiently
increase
heretofore
in rat
cytoplasmic
reported
pathways
hepatocytes,
free
calcium
leading
to c-myc
we found
that
concentrations,
gene
isoflavonoids a finding
not
previously. MATERIALS
AND METHODS
Preparation parenchymal cells: Parenchymal cells were isolated from --of liver male Sprague-Dawley or Wistar rats, weighing 200 g, using the collagenase perfusion method of Berry and Friend (11). Cells were suspended in modified Hanks' solution containing (in mM): NaC1,137; KC1,3.5; KHzPOr,0.44; NaHC03,4.2; NazHPOr,0.33; CaC12,l.O; Hepes/NaOH (pH 7.4),20; equilibrated with 02 as. In some experiments, extracellular calcium was reduced to 1 uM using Ca P+ -Ethylene Glycol Bis-(B-aminoethyl ether) N,N,N',N'-Tetraacetic Acid (EGTA) buffer (12). Measurement of cytoplasmic calcium concentration & aequorin: Aequorin was loaded into hepatocytes by making plasma membrane reversibly permeable using the method of Borle et al. (13). The cells were washed twice with ice-coldCa2+-free phosphate-buffered saline (in mM: NaC1,135; KC1,4; KHzPOs,0.51; glucose,ll; pH 7.4). and once with the same buffer containing 1-mM EGTA for 30 sec. Between each wash, the cells were centrifuged at 50 g for 2 min and the supernatant was discarded. An aliquot of packed cells was then suspended and incubated for 10 min at 4'C in 0.5 ml of a buffered salt solution containing 10 ug of aequorin (in mM: NaC1,140; Hepes,3; pH 7.4). Finally, the cells were centrifuged at 200 g for 30 sec. The supernatant was discarded. Aequorin-loaded hepatocytes were incubated in modified Hanks' solution. A cell suspension containing lo7 aequorin-loaded cells in 1 ml was put into a cuvette and incubated at 37'C under constant stirring. The aequorin signal was measured by using a Chrono-log platelet aggregometer (Havertown, PA) as described previously (14). Chemicals Aequorin was purchased from Dr. J. R. Blinks, Mayo Foundation (Rochester, MN). Genistein was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA) Psi-tectorigenin and orobol were kindly provided by Dr. T. Takeuchi, The Institute of Medical Chemistry, Tokyo, Japan. All of these compounds were dissolved in dimethylsulfoxide. The concentration of dimethylsulfoxide in the incubation buffer was 0.1%. RESULTS We attempted genistein, furathe
to
assess
psi-tectorigenin loaded
hepatocytes,
fluorescence
cytoplasmic
by these free
calcium
the
effect
and orobol but
without
of the
isoflavonoid
on cytoplasmic success
compounds, free
calcium
of
interference
because
compounds.
We then
examined
by measuring
changes
in aequorin
895
the
effects luminescence
using of on using
Vol.
BIOCHEMICAL
182, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a
lrni”
1 min
Figure 1. Effect of isoflavonoids on cytoplasmic free calcium. Aequorinloaded hepatocytes were stimulated by 50 ug/ml of genistein (a), orobol (b), or psi-tectorigenin (c) as indicated by arrows in the presence of 1-mM extracellular calcium.
loaded hepatocytes.
aequorin
transiently
increased
were incubated significant
cytoplasmic
in buffer
action
period
free
containing
was elicited
1,000 nM and the calcium a shorter
Surprisingly,
all
calcium concentration
1 mM calcium
by genistein;
transient
of the isoflavonoids
lasted
(Fig.
when the cells
1).
The most
the peak value was 800 -
for 2 min.
of the response was observed with
A lower magnitude
the addition
and
of orobol
and psi-tectorigenin. The elevation might relate integrity
in cytoplasmic
to possible
of the cell
massive calcium
result
suggested
the integrity
the presence of genistein. be the effect the calcium intracellular
treatment
transient
on a physiological
transient
regulation
cation
896
ionophore, (Fig.
2).
A A23187 This
membrane was maintained by genistein
of cytoplasmic
channel at the plasma membrane or calcium stores.
We examined the
with genistein.
by genistein
of the cell
The calcium
by the isoflavonoids
membrane.
was observed when divalent
the calcium that
concentration
damage of the cell
membrane following
influx
was added following
calcium
in
appeared to
calcium
mobilization
such as from
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
1
RESEARCH COMMUNICATIONS
c
10,000
: 5
1,000
180 l_l_-___l\i_
1 mln
Figure 2. Comparison of the effect of genistein and A23187 on cytoplasmic free calcium. Aequorin-loaded hepatocytes were stimulated with 50 ug/ml genistein (first arrow), following which 1 UM of A23187 was added (second arrow).
To identify
the source of calcium
loaded hepatocytes
were incubated
the calcium
through
influx
A smaller
(12). elicited
is intracellular also involved
in cytoplasmic
(Fig.
Calcium influx
in the calcium
aequorinmedium, where
channel is expected
increase
of genistein
stores.
by genistein,
in 1 uM calcium-containing
the calcium
but significant
by the addition
mobilized
to be negligible calcium was
3), hence the source of calcium
from extracellular
transient
of
spaces might be
in the medium containing
1 mM
calcium.
DISCUSSION We obtained tectorigenin, This effect
evidence that
and orobol
calcium
of fura-
elevate
has not heretofore
intracellular
or quin-2
the isoflavonoids,
signal
cytoplasmic been reported
is usually
cannot be explained
kinase
(1,2,6),
by other
PI turnover
(2-6),
free calcium probably
psiconcentrations.
because the
measured by monitoring
which is interfered
action
genistein,
fluorescence This
with by some isoflavonoids.
actions
including
topoisomerase
II
inhibition (7-9)
of tyrosine
and S6 kinase
(10). Ligand coupled activation inositol
trisphosphate
from an intracellular isoflavonoids
of PI turnover
which activate store
is contrary
(2-6).
protein The calcium
to the finding 897
that
produces diacylglycerol kinase
and
C and mobilize
mobilizing isoflavonoids
effect inhibit
calcium of agonist
Vol.
182,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
500-
2 c V
L
150
1 min
Figure 3. Effect of genistein on cytoplasmic free calcium in the presence of I-uM extracellular calcium. Aequorin loaded hepatocytes were stimulated with 50 ug/ml of genistein (arrow) in the presence of 1 UM extracellular calcium. The concentration of calcium was fixed by using Ca'+-EGTA buffer.
activation
of
platelets
(4,5).
cell
PI turnover
system.
The effect
suggest
generation
measured
Therefore,
the
more complex
than
the
using
by which
ongoing
fibroblasts
to
calcium
isoflavonoids isoflavonoids
with
of
inositol
phosphate
decrease
of
Li+
(manuscript
In addition, handling.
these
is
compounds
to be evaluated cellular
rat
metabolism
Our present
mobilize
the
in preparation).
on phosphoinositide
need
differs
cultures
not
date.
and
primary
did
presence
(3,6),
metabolism
done using
of isoflavonoids
cellular
(2),
on phosphoinositide
genistein
considered
obtained
of
in
affect
The mechanism subject
that
effect
independently data
A431 cells
Our investigations
hepatocytes
that
in
calcium
results with
suggest
caution. remains
the
study.
ACKNOWLEDGMENTS We thank Dr. Takashi Terano for advice and Yumiko Sugiyama for technical assistance. This work was supported by a Grant-in-Aid from the for a Comprehensive lo-Year strategy Ministry of Health and Welfare Japan, for Cancer Control, and by a grant from Kanae Foundation.
REFERENCES 1. Akiyama, T., N -, Shibuya,
Ishida, J., Nakagawa, S., Ogawara, M. and Fukami, Y. (1987) J. Biol.
898
H., Watanabe, S., Itoh, Chem. 262, 5592-5595.
Vol.
182, No. 2, 1992 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Imoto, M., Yamashita, T., Sawa, T., Kurasawa, S., Naganawa, H., Takeuchi, T., Bao-quan, 2. and Umezawa, K. (1988) FEBS Lett. 230, 43-46. Dean, N.M., Kanemitsu, M. and Boynton, A.L. (1989) Biochem. Biophys. Res. Commun. 165, 795-801. Gaudette, D.C. and Holub, B.J. (1990) Biochem. Biophys. Res. Commun. 170, 238-242. A.K. and Shukla, S.D. (1990) Mol. Pharmacol. 37, 519Dhar, A., Paul, 525. Hill, T.D., Dean, N.M., Mordan, L.J., Lau, A.F., Kanemitsu, M.Y. and Boynton, A.L. (1990) Science 248, 1660-1663. Okura, A., Arakawa, H., Oka, H., Yoshinari, T. and Monden, Y. (1988) Biochem. Biophys. Res. Commun. 157, 183-189. Yamashita, Y., Kawada, S. and Nakano, H. (1990) Biochem. Pharmacol. 39, 737-744. Markovits, J., Linassier, C., Fosse, P., Couprie, J., Pierre, J., Sablon, A.J., Saucier, J.M., Pecq, J.B.L. and Larsen, A.K. (1989) Cancer Res. 49, 5111-5117. Linassier, C., Pierre, M., Pecq, J.B.L. and Pierre, J. (1990) Biochem. Pharmacol. 39, 187-193. Berry, M.N. and Friend, D.S. (1969) J. Cell Biol. 4, 506. F. and Claret, M. (1984) Biochem. Mauger, J.P., Poggioli, J., Guesdon, J. 221, 121-127. Borle, A.B., Freudrich, C.C. and Snowdowne, K.W. (1986) Am. J. Physiol. 251, C323-C326. Mine, T., Kojima, I. and Ogata, E. (1990) Endocrinology 126, 2831-2236.
899
ISOFLAVONOIDS,
Takeshi
Tomonaga', Tetsuya Mine', Haruyuki Hayashi'**, of
'Surgery Medicine,
Department of Medicine,
31nstitute
Received
AND BIOPHYSICAL
GENISTEIN, PSI-TECTORIGENIN, FREE CALCIUM IN ISOLATED
Departments 'Fourth
BIOCHEMICAL
RESEARCH COMMUNICATIONS Pages 894-899
AND OROBOL, INCREASE CYTOPLASMIC RAT HEPATOCYTES
Itaru Kojima3, Masanori and Kaichi Isono'
and 4Biochemistry, Chiba University 1-8-1 Inohana, Chiba 280, Japan
School
of
of Internal Medicine, University of Tokyo School 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan
of Endocrinology, Gunma University, 3-39-15 Maebashi, Gunma 371, Japan
December
Taira4*,
10,
Showa-machi,
1991
psi-tectorigenin and orobol have SUMMARY: Isoflavonoid compounds, genistein, been implicated as inhibitors of tyrosine-specific protein kinase and phosphatidylinositol turnover. These compounds have been frequently used as a pharmacological tool to assess signal transduction pathways in various cell systems. In the course of analyzing signaling pathways in rat hepatocytes, we obtained an unexpected finding that these corn ounds transiently increase cytoplasmic free calcium. Since the CaZP mobilizing effect was observed in 1 uM calcium containing buffer, the source of the Ca2+ may be intracellular stores. Thus, when interpreting data obtained 0 1992 Academic Press, Inc. using these compounds, caution is needed.
Multi-step factor
signaling
activation
Inhibitors molecular
of
of each
inhibitor
in
of isoflavonoids,
including (PI)
involved receptor
signaling
in
compound,
tyrosine-specific
are
surface
involved
of
phosphatidylinositol
cell
component
mechanisms
An isoflavonoid
the
events
the
protein
transduction
leading
to
of
nuclear
should
aid
growth
events.
in elucidating
transduction. was first
kinase
psi-tectorigenin turnover
the
pathways
signal
genistein
in
was also
(1).
designated The inhibitory
and orobol, reported
as an
(2-6).
effects
on Other
workers
*Present address: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. *qo whom correspondence should be addressed. Abbreviations:PI, phosphatidylinositol; EGTA, Ethylene Glycol Bis-(Baminoethyl ether) N,N,N',N'-tetraacetic Acid. ooo6-291x/92 Copyright All rights
$1.50
0 1992 by Academic Press, of reproduction in any form
Inc. resewed.
894
Vol.
182, No. 2, 1992
noted
the
These
compounds
pathways
BIOCHEMICAL
inhibition
even
of have
though
AND BIOPHYSICAL
topoisomerase
II
been frequently specificity
(7-9)
used of
the
or 56 kinase
to assess
action
RESEARCH COMMUNICATIONS
of
signal these
(10)
activity.
transduction compounds
is
not
clear. In the expression
course
of analyzing
signaling
and DNA synthesis
transiently
increase
heretofore
in rat
cytoplasmic
reported
pathways
hepatocytes,
free
calcium
leading
to c-myc
we found
that
concentrations,
gene
isoflavonoids a finding
not
previously. MATERIALS
AND METHODS
Preparation parenchymal cells: Parenchymal cells were isolated from --of liver male Sprague-Dawley or Wistar rats, weighing 200 g, using the collagenase perfusion method of Berry and Friend (11). Cells were suspended in modified Hanks' solution containing (in mM): NaC1,137; KC1,3.5; KHzPOr,0.44; NaHC03,4.2; NazHPOr,0.33; CaC12,l.O; Hepes/NaOH (pH 7.4),20; equilibrated with 02 as. In some experiments, extracellular calcium was reduced to 1 uM using Ca P+ -Ethylene Glycol Bis-(B-aminoethyl ether) N,N,N',N'-Tetraacetic Acid (EGTA) buffer (12). Measurement of cytoplasmic calcium concentration & aequorin: Aequorin was loaded into hepatocytes by making plasma membrane reversibly permeable using the method of Borle et al. (13). The cells were washed twice with ice-coldCa2+-free phosphate-buffered saline (in mM: NaC1,135; KC1,4; KHzPOs,0.51; glucose,ll; pH 7.4). and once with the same buffer containing 1-mM EGTA for 30 sec. Between each wash, the cells were centrifuged at 50 g for 2 min and the supernatant was discarded. An aliquot of packed cells was then suspended and incubated for 10 min at 4'C in 0.5 ml of a buffered salt solution containing 10 ug of aequorin (in mM: NaC1,140; Hepes,3; pH 7.4). Finally, the cells were centrifuged at 200 g for 30 sec. The supernatant was discarded. Aequorin-loaded hepatocytes were incubated in modified Hanks' solution. A cell suspension containing lo7 aequorin-loaded cells in 1 ml was put into a cuvette and incubated at 37'C under constant stirring. The aequorin signal was measured by using a Chrono-log platelet aggregometer (Havertown, PA) as described previously (14). Chemicals Aequorin was purchased from Dr. J. R. Blinks, Mayo Foundation (Rochester, MN). Genistein was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA) Psi-tectorigenin and orobol were kindly provided by Dr. T. Takeuchi, The Institute of Medical Chemistry, Tokyo, Japan. All of these compounds were dissolved in dimethylsulfoxide. The concentration of dimethylsulfoxide in the incubation buffer was 0.1%. RESULTS We attempted genistein, furathe
to
assess
psi-tectorigenin loaded
hepatocytes,
fluorescence
cytoplasmic
by these free
calcium
the
effect
and orobol but
without
of the
isoflavonoid
on cytoplasmic success
compounds, free
calcium
of
interference
because
compounds.
We then
examined
by measuring
changes
in aequorin
895
the
effects luminescence
using of on using
Vol.
BIOCHEMICAL
182, No. 2, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a
lrni”
1 min
Figure 1. Effect of isoflavonoids on cytoplasmic free calcium. Aequorinloaded hepatocytes were stimulated by 50 ug/ml of genistein (a), orobol (b), or psi-tectorigenin (c) as indicated by arrows in the presence of 1-mM extracellular calcium.
loaded hepatocytes.
aequorin
transiently
increased
were incubated significant
cytoplasmic
in buffer
action
period
free
containing
was elicited
1,000 nM and the calcium a shorter
Surprisingly,
all
calcium concentration
1 mM calcium
by genistein;
transient
of the isoflavonoids
lasted
(Fig.
when the cells
1).
The most
the peak value was 800 -
for 2 min.
of the response was observed with
A lower magnitude
the addition
and
of orobol
and psi-tectorigenin. The elevation might relate integrity
in cytoplasmic
to possible
of the cell
massive calcium
result
suggested
the integrity
the presence of genistein. be the effect the calcium intracellular
treatment
transient
on a physiological
transient
regulation
cation
896
ionophore, (Fig.
2).
A A23187 This
membrane was maintained by genistein
of cytoplasmic
channel at the plasma membrane or calcium stores.
We examined the
with genistein.
by genistein
of the cell
The calcium
by the isoflavonoids
membrane.
was observed when divalent
the calcium that
concentration
damage of the cell
membrane following
influx
was added following
calcium
in
appeared to
calcium
mobilization
such as from
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
1
RESEARCH COMMUNICATIONS
c
10,000
: 5
1,000
180 l_l_-___l\i_
1 mln
Figure 2. Comparison of the effect of genistein and A23187 on cytoplasmic free calcium. Aequorin-loaded hepatocytes were stimulated with 50 ug/ml genistein (first arrow), following which 1 UM of A23187 was added (second arrow).
To identify
the source of calcium
loaded hepatocytes
were incubated
the calcium
through
influx
A smaller
(12). elicited
is intracellular also involved
in cytoplasmic
(Fig.
Calcium influx
in the calcium
aequorinmedium, where
channel is expected
increase
of genistein
stores.
by genistein,
in 1 uM calcium-containing
the calcium
but significant
by the addition
mobilized
to be negligible calcium was
3), hence the source of calcium
from extracellular
transient
of
spaces might be
in the medium containing
1 mM
calcium.
DISCUSSION We obtained tectorigenin, This effect
evidence that
and orobol
calcium
of fura-
elevate
has not heretofore
intracellular
or quin-2
the isoflavonoids,
signal
cytoplasmic been reported
is usually
cannot be explained
kinase
(1,2,6),
by other
PI turnover
(2-6),
free calcium probably
psiconcentrations.
because the
measured by monitoring
which is interfered
action
genistein,
fluorescence This
with by some isoflavonoids.
actions
including
topoisomerase
II
inhibition (7-9)
of tyrosine
and S6 kinase
(10). Ligand coupled activation inositol
trisphosphate
from an intracellular isoflavonoids
of PI turnover
which activate store
is contrary
(2-6).
protein The calcium
to the finding 897
that
produces diacylglycerol kinase
and
C and mobilize
mobilizing isoflavonoids
effect inhibit
calcium of agonist
Vol.
182,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
500-
2 c V
L
150
1 min
Figure 3. Effect of genistein on cytoplasmic free calcium in the presence of I-uM extracellular calcium. Aequorin loaded hepatocytes were stimulated with 50 ug/ml of genistein (arrow) in the presence of 1 UM extracellular calcium. The concentration of calcium was fixed by using Ca'+-EGTA buffer.
activation
of
platelets
(4,5).
cell
PI turnover
system.
The effect
suggest
generation
measured
Therefore,
the
more complex
than
the
using
by which
ongoing
fibroblasts
to
calcium
isoflavonoids isoflavonoids
with
of
inositol
phosphate
decrease
of
Li+
(manuscript
In addition, handling.
these
is
compounds
to be evaluated cellular
rat
metabolism
Our present
mobilize
the
in preparation).
on phosphoinositide
need
differs
cultures
not
date.
and
primary
did
presence
(3,6),
metabolism
done using
of isoflavonoids
cellular
(2),
on phosphoinositide
genistein
considered
obtained
of
in
affect
The mechanism subject
that
effect
independently data
A431 cells
Our investigations
hepatocytes
that
in
calcium
results with
suggest
caution. remains
the
study.
ACKNOWLEDGMENTS We thank Dr. Takashi Terano for advice and Yumiko Sugiyama for technical assistance. This work was supported by a Grant-in-Aid from the for a Comprehensive lo-Year strategy Ministry of Health and Welfare Japan, for Cancer Control, and by a grant from Kanae Foundation.
REFERENCES 1. Akiyama, T., N -, Shibuya,
Ishida, J., Nakagawa, S., Ogawara, M. and Fukami, Y. (1987) J. Biol.
898
H., Watanabe, S., Itoh, Chem. 262, 5592-5595.
Vol.
182, No. 2, 1992 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Imoto, M., Yamashita, T., Sawa, T., Kurasawa, S., Naganawa, H., Takeuchi, T., Bao-quan, 2. and Umezawa, K. (1988) FEBS Lett. 230, 43-46. Dean, N.M., Kanemitsu, M. and Boynton, A.L. (1989) Biochem. Biophys. Res. Commun. 165, 795-801. Gaudette, D.C. and Holub, B.J. (1990) Biochem. Biophys. Res. Commun. 170, 238-242. A.K. and Shukla, S.D. (1990) Mol. Pharmacol. 37, 519Dhar, A., Paul, 525. Hill, T.D., Dean, N.M., Mordan, L.J., Lau, A.F., Kanemitsu, M.Y. and Boynton, A.L. (1990) Science 248, 1660-1663. Okura, A., Arakawa, H., Oka, H., Yoshinari, T. and Monden, Y. (1988) Biochem. Biophys. Res. Commun. 157, 183-189. Yamashita, Y., Kawada, S. and Nakano, H. (1990) Biochem. Pharmacol. 39, 737-744. Markovits, J., Linassier, C., Fosse, P., Couprie, J., Pierre, J., Sablon, A.J., Saucier, J.M., Pecq, J.B.L. and Larsen, A.K. (1989) Cancer Res. 49, 5111-5117. Linassier, C., Pierre, M., Pecq, J.B.L. and Pierre, J. (1990) Biochem. Pharmacol. 39, 187-193. Berry, M.N. and Friend, D.S. (1969) J. Cell Biol. 4, 506. F. and Claret, M. (1984) Biochem. Mauger, J.P., Poggioli, J., Guesdon, J. 221, 121-127. Borle, A.B., Freudrich, C.C. and Snowdowne, K.W. (1986) Am. J. Physiol. 251, C323-C326. Mine, T., Kojima, I. and Ogata, E. (1990) Endocrinology 126, 2831-2236.
899
