- Email: [email protected]
Ca2+ Release by inositol trisphosphate from Ca2+-transporting microsomes derived from uterine sarcoplasmic reticulum
Vol.
130,
August
No. 3, 1985 15,
Ca2+
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1985
Pages
1027-1031
RELEASE BY INOSITOL TRISPHOSPHATE FROM Ca2+-TRANSPORTING MICROSOMES DERIVED FROM UTERINE SARCOPLASMIC RETICULUM Mary
Departments School Received
July
E. Carsten
of Obstetrics of Medicine,
and Jordan
D. Miller
and Gynecology and Anesthesiology UCLA, Los Angeles, California
1, 1985
Summarv: Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 uM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that trisphosphate may be by mobilizing intracellular Ca2+, inositol the link between hormonal stimuli and smooth muscle contraction. 0 1985
Academic
Press,
In
smooth
increased internal It
is
Inc.
not
muscle,
receptor
free
calcium,
stores
(l),
most
likely
what
causes
the
known
sarcoplasmic
reticulum.
laboratories
it
has
phosphatidylinositol directly While
contraction
originating
at
basis
suggested
others
calcium
have
muscles
looked
(3,4),
from at
the
least
release
On the been
is in
to
calcium
of
work the
of
from in
the
various of
trisphosphate
intracellular
release
from
hydrolysis
inositol
stores
phosphoinositide
on
reticulum.
of
that
based part
sarcoplasmic
4,5-bisphosphate
releases
smooth
induced
turnover
calcium
is
not
(2). in
some
as well
documented. Increased has (5).
more
been
free
demonstrated
However,
in
isolated
advantageous
biochemical
calcium
studies.
stimulated
by inositol
saponin-treated
coronary
sarcoplasmic than
saponin
Although
trisphosphate
reticulum treated
saponin
artery
preparations preparations
treatment
cells are for
preferentially 0006-291X/85 $1.50
1027
All
Copyright 0 1985 righrs of reproduction
by Academic Press, Inc. in any form reserved.
Vol.
130,
No. 3, 1985
alters
cell
BIOCHEMICAL
membrane
permeability,
dependent
on
reticulum, concentration, occur
AND
and
it of
time
changes
BIOPHYSICAL
in
RESEARCH
also
COMMUNICATIONS
affects
exposure,
a variety
sarcoplasmic
temperature of
enzyme
and
activities
(6). The
experiments
calcium
reported
release
characterized uterine
by
evidence
that
intracellular
this
inositol
microsomal sarcoplasmic
in
derived
reticulum.
Our
in
cell
from
findings
trisphosphate
membranes
demonstrate
trisphosphate
fraction
inositol
communication
from
a well
pregnant
bovine
offer
releases free
the
first
calcium
from
of
smooth
preparations
muscle. Materials
and Methods
Uteri, obtained at the slaughterhouse from close-to-term pregnant cows, were immediately dissected and the myometrium carefully stripped free of endometrium as described (7). The muscle strips were rinsed, immersed in ice-cold buffer (0.3 M sucrose, 0.01 M glucose, 5 mM dithiothreitol, 0.02 M Tris, pH and transported on ice to the laboratory. The muscle 7.2), tissue was diced with scissors, minced in a meat grinder, and homogenized in a Waring Blender first for 15 s, and again for All operations were carried out in the cold room at 10 s. 0 to 4oc. Differential centrifugation was at 2,500g for 20 min, 15,000g for 20 min. in a Sorvall GS-3 rotor and at 40,OOOg for 90 min.in a Spinco 21 rotor The final pellets were suspended in 0.08 M NaCl, 0.005 M sodium oxalate and placed on a sucrose density gradient consisting of layers of 35,45, and 55% sucrose. After 3 h of centrifugation in a Spinco 27.1 swinging bucket (average force 63,OOOg), the main protein layer was rotor The isolated from the 35% sucrose layer (density of 1.136). protein was stored at 4OC and used the following day. Protein concentration was determined by the method of Lowry et al (8). Calcium uptake and release were determined by the filtration method (7). Free metal concentrations were calculated according to Fabiato and Fabiato (9). D-myo-inositol trisphosphate was obtained from Sigma. Results
and Discussion Experiments
to
exclude
calcium
uptake
to
nmol
15.8
ATP-dependent
were calcium
carried uptake
amounted Ca/mg
out
protein
Ca2+ uptake
to
by 13.6 at
in
the
presence
mitochondria. nmol
16 min.
was increased 1028
Ca/mg In to
of
Na azide
ATP-dependent protein
the 19
at
8 min.
and
presence
of oxalate
and 29.2
nmol
Ca/mg
Vol.
130,
BIOCHEMICAL
No. 3, 1985
AND
Time
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(minutes)
Ficr.1. Calcium release by inositol trisphosphate from derived from bovine myometrium. The microsomal vesicles, microsomes were allowed to take up calcium from a solution containing 100 mM XCl, 20 uM imidazole buffer, pH 7.0, 5 mM NaNa3, 2 mM MgC12, 20 uM CaCl2 containing 45Ca, 0.5 mg microsomal protein/ml in the presence and absence of 2 mM ATP. ATP-dependent calcium uptake is the difference between the two measurements. Incubation was at 37OC. At 8 min. 1 ml was filtered through a 0.4% Millipore filter and 2 mM EGTA was added. 1 min. later (Time 0) an.aliquot was removed and addition was made of 10 ul to give final concentration filtered, of ionophore A23107 0.2 uM, inositol trisphosphate (Ins P3) 5 uM Further aliguots were taken at 1, or 1 uM, or deionized water. Aliquots of the filtrates were counted in a 2, 4 min. scintillation system. l :calcium uptake: u: 0.2 uM A23187; A: 5 uM Ins P3; A: 1 uM Ins P3; 0. H20 control.
protein,
is
characteristic
observed to
increase
data
in
The
the time
Ethyleneglycol(EGTA) from the
of
sarcoplasmic
in
calcium
literature
outside
vesicles.
reticulum.
of
added
the
of
Ca2+
to bring
< 10e8 vesicles
after
As shown in
uptake
by oxalate
Quantitatively
accumulation
release
bis-(P-aminoethylether)
7 x 10m6 to
calcium
the
at
pH 7.0
corresponds
is
shown
in
(10).
course
2mM was added
agents
of
Stimulation
respectively.
the Fig.
N,N,N'N'-tetraacetic
down the
M,(9)
free
and to
(11).
originate
calcium 1029
calcium
remove
Additional
EGTA must 1 all
Fig.
bound calcium from
was released
1. acid
concentration calcium
from
released inside from
by the
inside
Vol. 130, No. 3, 1985
the
BIOCHEMICAL
vesicles
dependent
by release
highest
dose
of
used,
ionophore.
It
breakdown
of
experiments place
the
to
Similar
reported
that
Mg2*
the were of
of
stimulation
Ca2+ uptake.
the
calcium
However,
this
calcium
outside
of
sarcolemmal
saponin
The myometrium
has
contrast
following
criteria
sarcoplasmic a Ca,Mg-ATPase (13),
oxalate
absence
of
inside
well
used
sarcolemma,
lack
phosphorylation reticulum results
molecular of
calmodulin
(14).
These ATPase
presented
trisphosphate
in
our
on
inside
or
show that
from
its
of
bovine The
laboratory. origin
The
from
presence
100,000
to
lanthanum
on ATPase
distinguish
this
paper
support
calcium
from
(ll),
characteristic effect
a cell
of
110,000
Ca2+ uptake
from
1030
calcium
distinguish
results
Ca,Mg-ATPase
properties
releases
to
an intracellular
derived
ATP-dependent
or
due
action
on the
sarcolemma.
weight
by
vesicles.
weight
of
of
establish
a molecular
uptake
inhibition
from
5 p
inositol
was
in
to
(12).
1 and
calcium
present
here,
than
stimulation
a 130,000
both
sites
characterized
rather
with
the
used
were
reticulum
an
does not
or the
some
2 x 10m5 M in
release
cells
the
in
a physiological
binding
fraction
been
to
reticulum
Ca2+ came from
microsomal
sarcoplasmic
from
In
out
treated
sarcoplasmic
Ca2+ pool.
released
and not
in
Since
calcium
rule
for
was used
EGTA at
of
the
as the
trisphosphate
termination
not
released
the
inositol
observed
Ca2+
(EDTA)
trisphosphate.
does
with
required
Mg2+ to
as with
release
Work with
between
of
after
a dose
Therefore
free
effect
inositol
EGTA, of
the
obtained
was added
addition
is
acid
lower
1 shows
40 % as much
trisphosphate.
potentiate
trisphosphate
inositol
Fig.
trisphosphate;
releasing
inositol
results
The
A23187. by inositol
5 uM,
has been
concentrations
of
calcium
EGTA so as to
attempt
the
ionophore
ethylenediaminetetraacetic
of
uptake.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
membrane
the
ATPase the
view
intracellular
(15). that
Vol.
130,
BIOCHEMICAL
No. 3, 1985
stores
of
uterine
smooth
trisphosphate calcium trisphosphate binding, smooth
to by
may intracellular
BIOPHYSICAL
cause
the
indeed
calcium
ionophore be
calcium
RESEARCH
Micromolar
muscle.
suffice released
AND
the
link
COMMUNICATIONS
amounts efflux
of
of
A23187.
Thus
between
agonist
rise,
and
A.R.
(1972)
contraction
inositol
40% of
the
inositol receptor of
uterine
muscle.
References 1.
2. 3. 4.
Siegman,
M.J.,
and Gordon,
Am. J.
Physiol.
222,
1587-1615.
6.
Berridge, M.J. (1984) Biochem. J. 220, 345-360. Takenawa, T. (1982) Cell Calcium 2, 359-368. Akhtar, R.A., and Abdel-Latif, A.A. (1984) Biochem. J. 224, 291-306. Suematsu, E., Hirata, M., Hashimoto, T and Kuriyama, 481-485. H. (1984) Biochem. Biophys. Res. Commun. Iz’d, Molec. Physiol. 3, Kwan, C.Y., and Lee, R.M.K.W. (1984)
7.
Carsten,
5.
105-114.
M.E.,
and Miller,
J.D.
(1977)
J.
Biol.
Chem.
252,
1576-1581.
8. 9. 10. 11. 12. 13. 14. 15.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. &93, 265-275. Fabiato, A., and Fabiato, F. (1979) J. Physiol. Paris 75, 463. Godfraind, T., Sturbois, X., and Verbeke, N. (1976) Biochim. Biophys. Acta 455, 254-268. Carsten, M.E., and Miller, J.D. (1984) Gynecol. Obstet. Invest. 17, 73-83. Downes, C.P., Mussat, M.C., and Michell, R.H. (1982) Biochem. J. 203,169-177. Carsten, M.E., and Miller, J.D. (1984) Arch. Biochem. Biophys. 232, 616-623. Carsten, M.E., and Miller, J.D. (1985) Sot. Gyn. Invest. 32, 216. Wuytack, F., Raeymaekers, L., Verbist, J., De Smedt, H., and Casteels, R. (1984) Biochem. J. 224, 445,451.
1031
130,
August
No. 3, 1985 15,
Ca2+
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1985
Pages
1027-1031
RELEASE BY INOSITOL TRISPHOSPHATE FROM Ca2+-TRANSPORTING MICROSOMES DERIVED FROM UTERINE SARCOPLASMIC RETICULUM Mary
Departments School Received
July
E. Carsten
of Obstetrics of Medicine,
and Jordan
D. Miller
and Gynecology and Anesthesiology UCLA, Los Angeles, California
1, 1985
Summarv: Microsomes derived from pregnant uterine sarcoplasmic reticulum, isolated by differential and sucrose density gradient centrifugation, accumulates Ca2+ in the presence of ATP. Inositol trisphosphate caused release of this Ca2+, in a dose dependent manner. 40% of the Ca2+ that can be released by the ionophore A23187 was released by 5 uM inositol trisphosphate. Removal of Mg by EDTA prior to addition of inositol trisphosphate did not change the course of Ca2+ release. These results indicate that trisphosphate may be by mobilizing intracellular Ca2+, inositol the link between hormonal stimuli and smooth muscle contraction. 0 1985
Academic
Press,
In
smooth
increased internal It
is
Inc.
not
muscle,
receptor
free
calcium,
stores
(l),
most
likely
what
causes
the
known
sarcoplasmic
reticulum.
laboratories
it
has
phosphatidylinositol directly While
contraction
originating
at
basis
suggested
others
calcium
have
muscles
looked
(3,4),
from at
the
least
release
On the been
is in
to
calcium
of
work the
of
from in
the
various of
trisphosphate
intracellular
release
from
hydrolysis
inositol
stores
phosphoinositide
on
reticulum.
of
that
based part
sarcoplasmic
4,5-bisphosphate
releases
smooth
induced
turnover
calcium
is
not
(2). in
some
as well
documented. Increased has (5).
more
been
free
demonstrated
However,
in
isolated
advantageous
biochemical
calcium
studies.
stimulated
by inositol
saponin-treated
coronary
sarcoplasmic than
saponin
Although
trisphosphate
reticulum treated
saponin
artery
preparations preparations
treatment
cells are for
preferentially 0006-291X/85 $1.50
1027
All
Copyright 0 1985 righrs of reproduction
by Academic Press, Inc. in any form reserved.
Vol.
130,
No. 3, 1985
alters
cell
BIOCHEMICAL
membrane
permeability,
dependent
on
reticulum, concentration, occur
AND
and
it of
time
changes
BIOPHYSICAL
in
RESEARCH
also
COMMUNICATIONS
affects
exposure,
a variety
sarcoplasmic
temperature of
enzyme
and
activities
(6). The
experiments
calcium
reported
release
characterized uterine
by
evidence
that
intracellular
this
inositol
microsomal sarcoplasmic
in
derived
reticulum.
Our
in
cell
from
findings
trisphosphate
membranes
demonstrate
trisphosphate
fraction
inositol
communication
from
a well
pregnant
bovine
offer
releases free
the
first
calcium
from
of
smooth
preparations
muscle. Materials
and Methods
Uteri, obtained at the slaughterhouse from close-to-term pregnant cows, were immediately dissected and the myometrium carefully stripped free of endometrium as described (7). The muscle strips were rinsed, immersed in ice-cold buffer (0.3 M sucrose, 0.01 M glucose, 5 mM dithiothreitol, 0.02 M Tris, pH and transported on ice to the laboratory. The muscle 7.2), tissue was diced with scissors, minced in a meat grinder, and homogenized in a Waring Blender first for 15 s, and again for All operations were carried out in the cold room at 10 s. 0 to 4oc. Differential centrifugation was at 2,500g for 20 min, 15,000g for 20 min. in a Sorvall GS-3 rotor and at 40,OOOg for 90 min.in a Spinco 21 rotor The final pellets were suspended in 0.08 M NaCl, 0.005 M sodium oxalate and placed on a sucrose density gradient consisting of layers of 35,45, and 55% sucrose. After 3 h of centrifugation in a Spinco 27.1 swinging bucket (average force 63,OOOg), the main protein layer was rotor The isolated from the 35% sucrose layer (density of 1.136). protein was stored at 4OC and used the following day. Protein concentration was determined by the method of Lowry et al (8). Calcium uptake and release were determined by the filtration method (7). Free metal concentrations were calculated according to Fabiato and Fabiato (9). D-myo-inositol trisphosphate was obtained from Sigma. Results
and Discussion Experiments
to
exclude
calcium
uptake
to
nmol
15.8
ATP-dependent
were calcium
carried uptake
amounted Ca/mg
out
protein
Ca2+ uptake
to
by 13.6 at
in
the
presence
mitochondria. nmol
16 min.
was increased 1028
Ca/mg In to
of
Na azide
ATP-dependent protein
the 19
at
8 min.
and
presence
of oxalate
and 29.2
nmol
Ca/mg
Vol.
130,
BIOCHEMICAL
No. 3, 1985
AND
Time
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(minutes)
Ficr.1. Calcium release by inositol trisphosphate from derived from bovine myometrium. The microsomal vesicles, microsomes were allowed to take up calcium from a solution containing 100 mM XCl, 20 uM imidazole buffer, pH 7.0, 5 mM NaNa3, 2 mM MgC12, 20 uM CaCl2 containing 45Ca, 0.5 mg microsomal protein/ml in the presence and absence of 2 mM ATP. ATP-dependent calcium uptake is the difference between the two measurements. Incubation was at 37OC. At 8 min. 1 ml was filtered through a 0.4% Millipore filter and 2 mM EGTA was added. 1 min. later (Time 0) an.aliquot was removed and addition was made of 10 ul to give final concentration filtered, of ionophore A23107 0.2 uM, inositol trisphosphate (Ins P3) 5 uM Further aliguots were taken at 1, or 1 uM, or deionized water. Aliquots of the filtrates were counted in a 2, 4 min. scintillation system. l :calcium uptake: u: 0.2 uM A23187; A: 5 uM Ins P3; A: 1 uM Ins P3; 0. H20 control.
protein,
is
characteristic
observed to
increase
data
in
The
the time
Ethyleneglycol(EGTA) from the
of
sarcoplasmic
in
calcium
literature
outside
vesicles.
reticulum.
of
added
the
of
Ca2+
to bring
< 10e8 vesicles
after
As shown in
uptake
by oxalate
Quantitatively
accumulation
release
bis-(P-aminoethylether)
7 x 10m6 to
calcium
the
at
pH 7.0
corresponds
is
shown
in
(10).
course
2mM was added
agents
of
Stimulation
respectively.
the Fig.
N,N,N'N'-tetraacetic
down the
M,(9)
free
and to
(11).
originate
calcium 1029
calcium
remove
Additional
EGTA must 1 all
Fig.
bound calcium from
was released
1. acid
concentration calcium
from
released inside from
by the
inside
Vol. 130, No. 3, 1985
the
BIOCHEMICAL
vesicles
dependent
by release
highest
dose
of
used,
ionophore.
It
breakdown
of
experiments place
the
to
Similar
reported
that
Mg2*
the were of
of
stimulation
Ca2+ uptake.
the
calcium
However,
this
calcium
outside
of
sarcolemmal
saponin
The myometrium
has
contrast
following
criteria
sarcoplasmic a Ca,Mg-ATPase (13),
oxalate
absence
of
inside
well
used
sarcolemma,
lack
phosphorylation reticulum results
molecular of
calmodulin
(14).
These ATPase
presented
trisphosphate
in
our
on
inside
or
show that
from
its
of
bovine The
laboratory. origin
The
from
presence
100,000
to
lanthanum
on ATPase
distinguish
this
paper
support
calcium
from
(ll),
characteristic effect
a cell
of
110,000
Ca2+ uptake
from
1030
calcium
distinguish
results
Ca,Mg-ATPase
properties
releases
to
an intracellular
derived
ATP-dependent
or
due
action
on the
sarcolemma.
weight
by
vesicles.
weight
of
of
establish
a molecular
uptake
inhibition
from
5 p
inositol
was
in
to
(12).
1 and
calcium
present
here,
than
stimulation
a 130,000
both
sites
characterized
rather
with
the
used
were
reticulum
an
does not
or the
some
2 x 10m5 M in
release
cells
the
in
a physiological
binding
fraction
been
to
reticulum
Ca2+ came from
microsomal
sarcoplasmic
from
In
out
treated
sarcoplasmic
Ca2+ pool.
released
and not
in
Since
calcium
rule
for
was used
EGTA at
of
the
as the
trisphosphate
termination
not
released
the
inositol
observed
Ca2+
(EDTA)
trisphosphate.
does
with
required
Mg2+ to
as with
release
Work with
between
of
after
a dose
Therefore
free
effect
inositol
EGTA, of
the
obtained
was added
addition
is
acid
lower
1 shows
40 % as much
trisphosphate.
potentiate
trisphosphate
inositol
Fig.
trisphosphate;
releasing
inositol
results
The
A23187. by inositol
5 uM,
has been
concentrations
of
calcium
EGTA so as to
attempt
the
ionophore
ethylenediaminetetraacetic
of
uptake.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
membrane
the
ATPase the
view
intracellular
(15). that
Vol.
130,
BIOCHEMICAL
No. 3, 1985
stores
of
uterine
smooth
trisphosphate calcium trisphosphate binding, smooth
to by
may intracellular
BIOPHYSICAL
cause
the
indeed
calcium
ionophore be
calcium
RESEARCH
Micromolar
muscle.
suffice released
AND
the
link
COMMUNICATIONS
amounts efflux
of
of
A23187.
Thus
between
agonist
rise,
and
A.R.
(1972)
contraction
inositol
40% of
the
inositol receptor of
uterine
muscle.
References 1.
2. 3. 4.
Siegman,
M.J.,
and Gordon,
Am. J.
Physiol.
222,
1587-1615.
6.
Berridge, M.J. (1984) Biochem. J. 220, 345-360. Takenawa, T. (1982) Cell Calcium 2, 359-368. Akhtar, R.A., and Abdel-Latif, A.A. (1984) Biochem. J. 224, 291-306. Suematsu, E., Hirata, M., Hashimoto, T and Kuriyama, 481-485. H. (1984) Biochem. Biophys. Res. Commun. Iz’d, Molec. Physiol. 3, Kwan, C.Y., and Lee, R.M.K.W. (1984)
7.
Carsten,
5.
105-114.
M.E.,
and Miller,
J.D.
(1977)
J.
Biol.
Chem.
252,
1576-1581.
8. 9. 10. 11. 12. 13. 14. 15.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. &93, 265-275. Fabiato, A., and Fabiato, F. (1979) J. Physiol. Paris 75, 463. Godfraind, T., Sturbois, X., and Verbeke, N. (1976) Biochim. Biophys. Acta 455, 254-268. Carsten, M.E., and Miller, J.D. (1984) Gynecol. Obstet. Invest. 17, 73-83. Downes, C.P., Mussat, M.C., and Michell, R.H. (1982) Biochem. J. 203,169-177. Carsten, M.E., and Miller, J.D. (1984) Arch. Biochem. Biophys. 232, 616-623. Carsten, M.E., and Miller, J.D. (1985) Sot. Gyn. Invest. 32, 216. Wuytack, F., Raeymaekers, L., Verbist, J., De Smedt, H., and Casteels, R. (1984) Biochem. J. 224, 445,451.
1031