Control of intracellular
in rat myometrium
Ca2+ activity
R, A. JANIS,
D. J. CRANKSHAW,
AND E. E. DANIEL
Department
of Pharmacology,
University
of Alberta, Edmonton,
Microsomal fractions from a variety of smooth muscles
have been isolated, including cow (7, 8), rat (3), and
human (2, 9) uterine smooth muscles, rabbit aorta (ZO),
and guinea pig intestine (26). However, the Ca2+-binding properties of these subcellular fractions have not
been studied with sufficient care at varying Ca2+ concentrations. Such a study is important if the physiological and pharmacological mechanisms by which the contractile state of smooth muscle is regulated are to be
elucidated.
In this study we have isolated subcellular fractions
enriched in plasma membrane (PM), smooth endoplasmic reticulum (SER), rough endoplasmic reticulum
(RER), and mitochondria from the myometrium of estrogen-dominated rats. We have studied the Ca2+-uptake characteristics of these fractions and have determined the influence of the free Ca2+ concentration upon
the amount of Ca2+ that can be taken up.
MATERIALS
AND
METHODS
Preparation of fractions. The method used for the
isolation of PM, SER, and RER is based on one previously described (29) for isolation of PM from rat myometrium. A number of modifications were employed to
increase the purity of PM and to isolate SER and RER
plasma membrane;
endoplasmic
reticulum;
mitochondria;
from the same sucrose gradient as PM.
Ca?+ transport; ionophores; Ca2+-ATPase
Female Wistar rats weighing 160-200 g were injected
subcutaneously with 200 ,ug diethylstilbestrol (dissolved
in peanut oil) daily for 2 days and were killed on the
REGULATIONOF THE INTRACELLULARCa2+ Concentration
third day by a blow on the head. The two uterine horns
is important for the control of the contractile and meta- were rapidly removed and placed in ice-cold 250 mM
bolic states of smooth muscle. An important step to- sucrose, 40 mM histidine solution at pH 7.0 (pH adjusted
wards a description of this regulation can be taken if one with HCl); this buffer solution was used for all subsecan determine which of the cellular organelles take part
quent steps unless otherwise stated. The uterine horns
in the control of Ca2+ movements. This problem-has
were trimmed of fat and loosely bound connective tisbeen approached experimentally
in several different
sue, slit open with scissors, and stripped of endomeways. Attempts have been made to study Ca2+ move- trium and most circular muscle; this entire process was
ments directly, by following the fluxes of %as+ in whole carried out on a filter paper moistened with buffer and
tissues of smooth muscle (31), and this technique has maintained at approximately 4°C on a cold plate (Therrecently been improved by the introduction of the CtLan- moelectrics Unlimited, Inc.). All subsequent operations
thanum Method” of van Breemen (48). However, the were carried out at 4°C unless otherwise stated. Myomerelevance of Ca2+ flux analyses to the elucidation of tria from 10 to 12 uteri were finely minced with scissors,
cellular Ca2+ movements in smooth muscle cells has suspended in 30 ml of buffer, and homogenized twice for
been severely
questioned (31), and doubt has been 10 s at 15,000 rpm with a Polytron pT20 (Kinematic
thrown
upon the applicability
of the Lanthanum
GMBH).
Method to uterine muscle (25).
Figure 1 shows the centrifugation procedures that
An alternate approach is to describe the Ca2+ uptake
were used. First, the homogenate was centrifuged at
of subcellular fractions enriched in certain organelles.
1,000 x g (avg) for IO min in order to remove nuclei,
C50
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
JANIS,
R. A., ID. J. CRANKSHAW,
AND E. E. DANIEL.
Control
of intracellular
Ca2+ activity in rat myometrium.
Am. J. Physiol. 232(l): C50--C58, 1977 or Am. J. Physiol: Endocrinol.
Metab.
Gastrointest.
Physiol.
I( 1): GO-C58,
1977. - Four
fractions enriched, respectively,
in plasma membrane
(PM),
smooth endoplasmic
reticulum
(SER), rough endoplasmic
reticulum (RER), and mitochondria
were isolated from estrogendominated
rat myometrium.
Ca2+ uptake by these fractions
was studied in order to estimate the relative potential
of the
corresponding
organelles for controlling
intracellular
Ca”+ activity. Ca2+ uptake properties
of the PM, SER, and RER
fractions were similar except that potentiation
by oxalate was
in the order RER 2 SER > PM. However, studies with the
ionophores X-537A aqd A23187 suggested that Ca2+ was transported into the lumen of membrane vesicles of all these fractions. Unlike that of skeletal muscle sarcoplasmic
reticulum,
Ca2+ uptake by the myometrial
fractions was not supported by
high-energy
compounds other than ATP. Mitochondria
took
up much less Ca”+ at low, and much more Ca2+ at high, free
Ca2+ concentrations
than did the other fractions. The amount
of Ca”+ taken up in 30 s from a 1 PM free Ca2+ solution in the
presence of ATP was similar for all fractions. These results
suggested that mitochondria
may act as an important
Ca”+
control system in rat myometrium
when the intracellular
Ca2+
concentration
is near 1 PM or higher, whereas the PM, SER,
and RER may be of major importance at Ca”+ levels of 0.3 PM
or lower.
Canada
CA'+
REGULATION
IN
RAT
c51
MYOMETRIUM
ml -l. As shown in Fig. 1, fractions designated PM, SER,
and RER were obtained from protein bands located at
the 8-B% interface, within the 33% sucrose band, and
Sediment
discarded
Supernatant
at the 33-45% interface, respectively.
70 min
Ertzymatic
determinations.
The following
were used
J
jl
as
plasma
membrane
marker
enzymes
(l&14):
Y-nucleSediment
is crude
Supernatant
otidase (44); K+-activated;
ouabain-sensitive
phosphamitochondrial
fraction
113,000xg
tase assayed as described by Kidwai and colleagues (29),
30 min
exceDt
that DH 7.4 and 1 mM ouabain were used; and
c
JI
phosbhodiesierase
I (46), Nonspecific
phosphastase
was
Sediment
Supernatant
discarded
estimated
using P-glycerolphosphate
and glucose 6112,OOOxg,720
min
sucrose
gradient
phosphate as substrates
(16). Nonspecific
phosphatase
activity was found TV be less than 10% of the 5’-nucleolO,OOOxg,lO
min
tidase activity in all fractions,
and therefore no correction for it was made.
Mg2+-ATPase
activities were measured in similar solutions to those used for Ca2+ uptake, except that all
ISER
Ca2+ was omitted, and 5 mM ATP and 0.15 mM ethyl*T (Sediment
is
Supernatant
RER
ene glycol-his-( P-aminoethyl
ether)JV,N’-tetraacetate
mitochondrial
fragments)
EGTA) were present.
Incubation
media for Ca2+ +
Mg2+-ATPase measurements
were the same as those for
FIG. 1. Flow
diagram
for isolation
of subcellular
fractions
enMg2+-ATPase,
except that 0.1 mM CaCl, was added.
riched in plasma
membrane
(PM),
smooth
endoplasmic
reticulum
The
ATPase
reactions
were started by the addition of 0.1
(SER), and rough endoplasmic
reticulum
(RER).
ml of protein (to give a final volume of 1 ml) and were
stopped after a lo-min incubation at 37°C by addition of
contractile proteins, whole cells, and unbroken tissue. 1 ml of cold 10% trichloroacetic acid (TCA). Inorganic
The supernatant from this centrifugation was centriphosphate was measured by the method of Fiske and
fuged at 10,000 x g (avg) for 10 min to yield a crude Subbarow (19) using 2,4-diaminophenol as the reducing
mitochondrial fraction (as the pellet) and a crude micro- agent (41). Protein was determined as described by
somal fraction (as the supernatant). This 10,000 x g
Lowry et al. (33), and cytochrome c oxidase activity was
centrifugation was omitted in some experiments as indimea&red by the method of Cooperstein and Lazarow
cated in Table 1.
(10)
Mitochondria to be used for studies of Ca2+ uptake
E&-on
microscopy. Pellets were fixed in buffered
glutaraldehyde (pH 7.1), postfixed in osmium tetroxide,
were then prepared by suspending the crude mitochondrial pellet in btier and centrifuging for 10 min at 1,000 and embedded in epon. Sections were stained with urax g (avg). The supernatant was centrifuged at 10,000 x nyl acetate and lead citrate, and photographed on a
g (avg) for 10 min, and the pellet was washed and resus- JEM 7A electron microscope. Several sections from each
pended in buffer. This latter centrifugation and wash- of several different preparations were examined.
ing was repeated once, and the final &ochondrial
pelMeasurement of Cu2+ uptake. Ca2+ uptake was mealet was resuspended in sucrose-histidine buffer to yield
sured using an adaptation of the filtration technique of
a uniform suspension containing l-5 mg protein ml-l.
Martonosi and Feretos (34) and was always done on
Teflon glass homogenizers were used for the suspension freshly prepared fractions. The reaction mixture conof all pellets.
tained: 100 mM KCl; 5 mM MgCl,; O-1 mM CaCl,,
The crude microsomal fraction was sedimented by labelled with 0,4 &i ml+ 4sCa2+;40 mM imidazole; and
centrifugation at 113,000 x g (avg) for 30 min, and the
lo-50 rug of protein per final milliliter of reaction mixture, depending on the fraction used and the conditions
pellet was suspended in 3 ml of btier. The resulting
suspension was carefully layered on the sucrose gra- of the assay. Where indicated in the text, 5 mM disodient. The gradient was composed of, from the bottom of dium ATP, 0.5 mM sodium azide or 5 mM potassium
the tube upwards, 4 ml of 45% sucrose and 3 ml each of oxalate, variable amounts of potassium EGTA, and var33.0 and 28.0% sucrose, respectively (Fig. 1). The su- ious substrates were added and dissolved directly in the
crose concentrations were checked with a refractometer.
incubation medium. The drug, X-537A and A23187,
The density gradient preparation was centrifuged at however, had to be added in an ethanol vehicle in such a
112,000 x g (avg) for 120 min in a swinging bucket rotor
way that the final concentration of ethanol in the incu(Beckman SW40) aft er which protein bands were care- bation medium became 1%. The reaction mixture was
fully removed from the gradient tube by Pasteur pi- bufFered at pH 7.0 with 40 mM imidazole that would
pettes. Individual fractions obtained were slowly di- prevent changes in the free Ca2+ concentration under
luted with btier and deionized distilled water to yield a our conditions due to changes in pH. Uptake reactions
final sucrose concentration of 8%. The resulting suspen- were carried out at 37°C in a shaking-water bath unless
sions were centrifuged at 10,000 x g (avg) for 10 min and otherwise indicated. Reactions were started by addition
the resulting supernatants were centrifuged at 102,000 of protein (0.1 ml for every 1 ml of final reaction mixx g (avg) for 30 min to yield the final pellets that were ture) accompanied by rapid mixing with a Vortex-Genie
used to prepare uniform suspensions of 0.2-l mg protein
after a preincubation time of 5 min at 37OC.The reaction
tlompgenate
Jl,OOOxg,lO
min
I 7o,oooxg,
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
c52
RESULTS
All of the protein from the sucrose gradient shown in
Fig. 1 was removed as 11 fractions. The enzymatic activities, Ca2+ uptake in the presence of azide, and morphology of these fractions were studied to determine which
fractions best represented plasma membrane, smooth
endoplasmic reticulum, and rough endoplasmic reticulum (unpublished studies). From these studies, fractions designated PM, SER, and RER (Fig. 1) were chosen for further study. The activities of these fractions
were compared with those of the 1,000 x g supernatant
(which was free of nuclei, collagen, and cells) and the
mitochondrial fraction isolated by differential centrifugation. Table 1 shows the enzymatic activities of these
fractions.
The PM exhibited a 14- to 45-fold enhancement in the
specific activity of plasma membrane marker enzymes
compared with the 1,000 x g supernatant. This fraction
had the highest specific activity of these enzymes and
the lowest cytochrome c oxidase activity. About 35% of
the total 5’-nucleotidase activity of the 1,000 x g supernatant was present in the PM, and another 35% was
present in bands between PM and SER (including their
10,000 x g sediments). The yield of PM was 0.37 mg
protein g-l wet wt of myometrium (n = 6), and PM
TABLE
1. Enzymatic
from rat myometrium
CRANKSHAW,
activities
Fraction
1,000 X g
Supernatant
5’-Nucleotidase
7.8t0.8
(10)
K+-stimulated,
ouabainsensitive
phosphatase
Phosphodiesterase
Cytochrome
I
c oxidase
DANIEL
of fractions
T
Enzyme
AND
0.07*0.13
(4)
0.120-t0.08
(7)
PM
111.523.4
(321
3.16~0.25
(61
SER
RER
28.Oe1.5
(30)
14.4k1.1
0.25*0.28
0.20-1-0.11
(61
1.67k0.54
(13)
0.393kO.58
l.OkO.2
13.5t1.4
(12)
(121
(121
(21)
(6)
0.253~0.14
(12)
29.923.6
(9)
Mitochondria
16.7k1.3
(1%
0.30~0.09
(41
0.151-+0.13
(31
31.4k3.7
(6)
Values
are means 2 SE. Specific
activities
are expressed
as micromoles
phosphate
except in the case of cytochrome
c oxidase activity
that
released milligram-’
protein hour-’
is expressed
as a percent of the specific activity
of the inner mitochondrial
membrane
fraction
obtained
from the sucrose gradient
tube; in these experiments
the 10,000 x g
centrifugation
before the sucrose gradient
step was omitted.
Numbers
in parentheses
are
the number of independent
preparations
tested.
contained 2.5% of the protein present in the 1,000 x g
supernatant. In comparison, the mitochondrial fraction
(designated mitochondria in Table 1) contained approximately twice as much protein as PM; SER and RER had
0.64% and 0.53% of the protein from the 1,000 x g
supernatant, respectively.
Representative electron micrographs of the PM, SER,
and RER are shown in Fig. 2. Triple layer membrane
structures were seen at higher magnifications of all
fractions (not shown). PM and SER consisted mainly of
vesicles not bounded by ribosomes, and RER was composed almost entirely of vesicles with attached ribosomes, Mitochondrial fragments were seen in the RER
fraction but not in PM or SER. The RER fraction was
designated as such on the basis of its appearance in all
electron micrograph examinations and its low contamination with plasma membrane marker enzymes (Table
1). The SER best represented material derived from
smooth endoplasmic reticulum because it consisted
mainly of smooth vesicles that exhibited low activity of
both plasma membrane and mitochondrial marker enzymes (Table 1). The lack of established marker enzymes for smooth muscle endoplasmic reticulum makes
this designation tentative.
Similarly, the extent to
which this fraction contaminated others is not known.
The specific activities of the plasma membrane marker
enzymes in the mitochondrial fraction isolated by differential centrifugation were 9-16% of those in the PM. In
addition to intact mitochondria, some smooth and rough
vesicles could be seen in electron micrographs of these
fractions (not shown). Some indication of the contamination of this fraction by SER and RER is obtained from
the effects of azide and of oxalate on Ca2+ uptake (Table
2). Nearly all of the Ca2+ uptake by the mitochondrial
fraction is azide sensitive, and the small increase in
Ca2+ uptake in the presence of oxalate is not statistically significant; both of these effects are in contrast to
those seen in the other fractions.
The lack of inhibition by azide in the PM (Table 2)
confirmed the results of Table 1 that indicated very
little mitochondrial contamination in this fraction. The
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
was stopped by withdrawing
0.8 ml with a Pipettman
(Gilson Medical Electronics,
Inc.) automatic pipette and
filtering through a 25-mm-diam
filter with pore size of
0.45 pm (Matheson-Higgins
Co.) that was held on a
Millipore 3025 filtration
manifold. Filtration
took 2-3 s
and was followed by a wash with 10 ml of 250 mM
sucrose-40 mM imidazole solution at pH 7.0 which took
8-12 s. Prior to use, all filters were washed with 10 ml of
100 mM KC1 solution followed by 10 ml of sucroseimidazole solution; this procedure was required for low
background counts.
Filters were dissolved in 10 ml of Bray’s solution (6)
and were counted to 2% accuracy. Appropriate blanks
(no protein added), controls, and aliquots of reaction
mixtures from blank tubes were counted for each experiment. No quench correction was required.
Free Ca++ concentration. The free Ca2+ concentration
in the reaction mixture was controlled by the use of
EGTA. The concentrations of EGTA required to produce
desired free Ca2+ concentrations were calculated from
the stability constants used by Godt (22) by means of the
equations of Katz et al. (30),
MateriaZs. Distilled water was deionized to a specific
resistance greater than 16 Ma Cmm2. Organic compounds were of the highest purity available from Sigma
Chemical Co. ; 4*5CaC1,was obtained from Amershaml
Searle Corp.
TerminoZogy . We have used Ca2+ uptake as an operational term to include all the processes whereby 45Ca2+
becomes associated with membrane vesicles. Ca2+ transport is used when we refer specifically to processes
leading to formation of a gradient of Ca2+ across the
vesicle membrane. We think the term binding should be
used for chemical association between Ca2+ and a membrane site.
JANIS,
CA'+
REGULATION
IN
RAT
MYOMETRIUM
c53
presence of some mitochondrial
membrane in SER and
RER is indicated by the azide sensitivity
of the Ca2+
uptake (Table 2) and by the cytochrome c oxidase activities (Table 1).
Because Ca2+ uptake by PM exhibits little potentiation by oxalate (Table 21, it appears that Ca2+ transport
might not take place in this fraction; alternatively,
the
vesicles might not be permeable to oxalate. Similarly,
the contribution
made by Ca2+ transport
to the total
uptake in SER and RER was unknown.
The results
obtained by Scarpa et al. (381, who were able to abolish
the Ca2+ gradient across membranes
with the ionophorous antibiotics
A23187 and X-537A, prompted us to use
these substances in the myometrial
system to determine
whether or not Ca2+ gradients are created. The presence
of 1% ethanol in the incubation medium had a negligible effect upon uptake by SER and RER although it
produced a slight inhibition of uptake by PM. The presence of both A23187 and X-537A rendered PM, SER, and
RER much less capable of retaining Ca2+ than they were
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
micrographs
of
FIG. 2. Electron
subcellular
fractions
enriched
in
plasma membrane
(PM), smooth endoplasmic
reticulum
(SER),
and rough
endoplasmic
reticulum
(RER).
c54
JANIS,
TABLE
2. Calcium
rat myometrium
Addition
tion
to IncubaMedium
uptake
by fractions
T-~
None
5 mM ATP
from
Fraction
PM
SER
5.2+1.0
5.OkO.6
4.9kl.O
RER
(6)
(6)
(6)
(6)
22.Oe2.7”
(12)
17.1*1.8*
(14)
16.9+1.2*
(10)
116.4?24*
5 mM ATP +
5 mM oxalate
29.2k3.6
(121
35.8*4.tH
(14)
48.328.9t
(10)
144.9-+30
5 mM ATP +
0.5 mM azide
21.9k3.0
11.9&0.8$
(14)
10.5?2.5$
(10)
7.4*1.3$
(12)
CRANKSHAW,
AND
DANIEL
The time courses for Ca2+ uptake by the three vesicular fractions (PM, SER, and RER) were found to be very
similar to each other (Fig. 3). They all showed very
rapid uptake from 17 PM free Ca2+ for the first 30 s and
reached steady-state
values of 20-30 pmol Ca2+ g-l protein in lo-15 min. In contrast, mitochondria
reached a
steady-state
value of about 100 pmol g-l protein in 5
of the
TABLE
4. ATPase activities
from rat myometrium
fractions
(121
Fraction
(12)
3. Effect of ionophores on Ca2+ uptake
from rat myometrium
by
Fraction
Additions
to Incubation
Medium
5 mM ATP
5 mM ATP + 1% ethanol
PM
SER
RER
23.2 k 5.0
17.5 k 2.9
12.8 k 2.5
17.2 + 2.4
14.5 k 3.3
17.3 * 3.5
5 mM ATP + 1% ethanol
A23187
t 10 pM
5.5 k 4.7
4.7 L 2.5
4.5 + 4.7
5 mM ATP
537A
+ 20 PM X-
7.5 k 2.5
9.8 k 4.2
4.1 * 1.2
+ 1% ethanol
Values are means + SE of the uptake of five preparations.
Ca*+ gram-’ protein.
Conditions
were as given in Table 2.
The units
RER
116 * 4
46 IL 3
34 k 3
MgATPase
104 k 5
41 2 3
26 k 1
12*
5
8*
are micromoles
in the absence of ionophore (Table 3). The residual Ca2+
uptake was similar to that in the absence of ATP (Table
2)
Table 4 shows the ATPase activities of the fractions.
In the absence of Ca 2+ the ATPase activity of PM was
250% greater than the’activity
of SER which was more
than 150% higher than the activity of RER. When Ca2+
was added, all fractions
showed increased splitting
of
ATP, but only that by PM and RER was shown to be
significant.
Although the enzyme responsible for the Ca2+ transport in skeletal muscle sarcoplasmic
reticulum
(SR) has
Deen termed an ATPase, it has been clearly shown that
inosine, guanosine,
cytidine,
or uridine triphosphate
(ITP, GTP, CTP, or UTP, respectively)
(24, 34), acetyl
phosphate (12), and paranitrophenyl
phosphate (28) can
provide the energy for transport
of Ca2+ in this system.
Therefore we determined
whether
Ca2+ uptake by the
myometrial
systems had a similar or different substrate
specificity.
Table 5 shows that none of the alternative
substrates
that was used was capable of elevating Ca2+
uptake by PM, SER, or RER above that occurring in the
absence of any substrate.
In fact, paranitrophenyl
phosphate decreased Ca2+ uptake by reticulum
fractions (P
< .05). When these experiments
were repeated with 5
mM oxalate present in the incubation
medium, essentially similar results were obtained (results not shown).
Difference
caused
= extra
by added
splitting
Ca2+
Values
are means
+ SE of the activities
of four preparations
expressed
as micromoles
Pi released
per milligram
protein.
Assay
conditions
are given in the text.
* The difference
is significant
at
P < 0.05 using a paired
Student
t test.
TABLE
5. Ca2+ uptake by subcellular
fractions
myometrium
in presence of various substrates
Additions
Fraction
to Incubating
Medium
None
5 mM
5 mM
5 mM
5 mM
5 mM
ATP
GTP
ITP
AcP
PNpP
of rat
PM
SER
RER
35*
5
100
21*
5
264
6
272
8
25 * 19
362
3
100
28*
7
324
7
26 2 11
21*
4
30*
3
100
16 t 10
30+
5
25 2 11
ll*
3
Values are means k SE from 3 preparations
and are expressed
as
a percent
of Ca2+ uptake
in the presence
of 5 mM ATP. Conditions
were as given in text.
30
l
50
5
Time
FIG. 3. Time
myometrium.
MgCIB, 0.1 mM
In upper curve
errors of means
and 15 min at
mitochondria.
IO
(min)
course of Ca2+ uptake
by subcellular
fractions
of rat
Incubation
medium
contained
5 mM ATP,
5 mM
CaCl,,
100 mM KCI, and 40 mM imidazole
at pH 7.0.
A represents
RER, l PM, and 0 SER. Standard
were less than 4 in all cases except for RER at 5.10
which points they were less than 12. Lower curve is
Points are means from 6 experiments
in all cases.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
TABLE
SER
Ca2+ + Mg2+ ATPase
(12)
Values are means t SE. The units are micromoles
Ca2+ gram-l protein for an incubation of 10 min at 37°C. The reaction mixture
contained
0.1 mM CaCl,, 5 mM MgC12, 100 mM
KCl, and 40 mM imidazole
at pH 7.0. Numbers
in parentheses
are the number of independent preparations
tested.
* Placed above values significantly
(P < 0.001) different
from
controls
without
ATP.
t Placed above values significantly
different
(P < 0.05) from
preparations
incubated
with ATP but without oxalate; all were significantly
different
from
controls
without
ATP.
$ Uptake
values
significantly
inhibited
by azide,
P <
0.01.
9 Uptake v a 1ue significantly
inhibited
by azide, P < 0.001.
fractions
PM
CA2+ REGULATION
IN
RAT
MYOMETRIUM
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
min under the same conditions.
The effects of the free
Ca2+ concentration
upon Ca2+ uptake were studied using incubation times of 10 min. Under these conditions,
less than 15% of the ATP present was hydrolyzed,
and
the amounts of calcium taken up could be accurately
measured by our technique.
Just as the time courses for Ca2+ uptake by PM, SER,
and RER were similar, so the dependences of Ca2+ uptake on the free Ca2+ concentration
were similar for
these three fractions
(Fig. 4). Mitochondria, however,
unlike the vesicular fractions, did not exhibit ATPdependent uptake from solutions of 0.03 of 0.3 PM free
Ca2+, but bound far more Ca2+ at 1 PM (Fig. 4) and
higher Ca2+ concentrations (Fig. 5) than did the other
fractions.
In order to compare the initial rates of Ca2+uptake by
the PM and mitochondrial fractions, total and ATPdependent binding by these fractions was measured
Coicium concentration
( JIM )
after a 30-s incubation at 20°C. The free Ca2+concentraFIG. 5. Effect
of Ca2+ concentration
on Ca2+ uptake
by mitochontion in these experiments (1 PM) was chosen to be
drial fraction
from rat myometrium.
Conditions
are same as for Fig.
similar to the intracellular Ca2+ activity of maximally
4, except
for change
in scale to demonstrate
full extent
of Ca2+
contracted myometrium (see DISCUSSION).
Total Ca2+ uptake
by this fraction
over range studied.
uptake was 1.3 t 0.2, pmol g-l protein for PM, and
0.7 5 0.2 pmol g-l for mitochondria, whereas ATPindependent uptake was 0.5 pmol g-l for both PM and and mitochondria in red skeletal and heart muscle (32,
mitochondria. Three independent observations were 42), but the work of Scarpa and Graziotti (39) indicates
made in all cases.
that Ca2+ uptake by mitochondria is grossly inadequate
for the regulation of the beat-to-beat Ca2+ cycle of mamDISCUSSION
malian heart. It is established that the plasma membrane of nerve (1), kidney (36), and erythrocytes (40) can
Intracellular Ca2+ activity of cells must be carefully
transport Ca2+, although the role of this organelle in
controlled because Ca2+ plays a key regulatory role in a
lowering intracellular Ca2+ after contraction of skeletal
variety of cellular processes (32). In muscle cells, Ca2+
(45) and smooth muscle (26) is not well established
enters or is released from internal stores to initiate
because it is not known if these preparations can transcontraction and must be removed to allow relaxation.
port Ca2+ from a solution of low Ca2+ activity at a
There is general agreement that the SR is the major
reasonable rate. In smooth muscle, no internal organregulator of Ca2+ activity during contraction in white
elle exists similar to the sarcoplasmic reticulum of
skeletal muscle (27), whereas mitochondria may be the
striated muscle in organization or in amount of memmost important system in cells such as those of liver,
brane. It has been suggested (5, 43) that mitochondria
kidney (4) and nerve (1) that do not have to remove
and endoplasmic reticulum play important roles in reglarge quantities of the ion so rapidly, There is some
ulation of intracellular Ca2+in vascular smooth muscle.
dispute as to the relative regulatory contributions of SR
Alternately,
an outwardly directed pump for Ca2+ located in the plasma membrane has been suggested as a
PM
SER
major mode of Ca2+ regulation in myometrium (49).
I
3or
In this study we have isolated membrane fractions
enriched in plasma membrane, simultaneously with
fractions somewhat enriched in smooth and rough endoplasmic reticulum and mitochondria from the loigitudinal muscle of rat uterus. Furthermore, we have investiMitochydria
gated the ability of these fractions to remove Ca2+ from
solutions of physiological Ca2+ concentrations,
The PM fraction that we isolated was at least 14 times
enriched in plasma membrane marker enzymes as compared to the 1,000 x g supernatant, and this represents
001 0.1
#oncentro tion ( pM)
a considerable improvement in purity over the plasma
membrane fraction from this tissue previously isolated
FIG. 4. Effect
of Ca2+ concentration
on Ca2+ uptake by subcellular
by Kidwai et al. (29). Table 1 shows that the SER and
fractions
of rat myometrium.
Incubation
medium
contained
5 mM
ATP, 5 mM MgCl,,
100 mM KCl, variable
amounts
of CaCl,
and
RER fractions had very low plasma membrane marker
EGTA,
and 40 mM imidazole
at 7.0 (open symbols),
Lower
trace
activity; however, contamination of these two fractions
(closed symbols)
shows Ca’ l+ binding
in absence of ATP. Incubations
by
mitochondrial material was quite substantial. SER
were for 10 min. Points shown are means of results from between
7
had 42% of its ATP-dependent Ca2+ uptake inhibited by
and 18 preparations.
Vertical
bars are 2 times
standard
error
of
mean.
the presence of azide (Table 2) and 42% of the specific
C56
JANIS,
7
AND
DANIEL
ATPase is responsible
concept that a Ca 2+-stimulated
for the Ca2+ transport
by these fractions. CJ.oser examination of the data reveals that such support is at best
very tenuous. Under the conditions
of ATPase assay
that we used, the free Ca2+ in the medium upon addition
of Ca2+ was 1 PM (30, 22). From a 1 PM Ca2+ solution,
PM, SER and RER all take up approximately
8 pmol
Ca2+ g-l protein in 10 min (Fig. 4). Thus if the stoichiometry that pertains in SR of skeletal muscle, whereby
two Ca2+ ions are transported
while one ATP molecule
is hydrolized (27), pertains also in PM, SER, and RER,
we should expect to see only 0.004 pmol Pi mg-l protein
released under these conditions.
Clearly such a small
amount is not measurable with the techniques we have
used. The much larger amount of Pi released in response
to added Ca2+ may indicate that a large proportion of the
ATPase normally responsible for Ca2+ transport
in PM,
SER, and RER has become “uncoupled”
or that there
exist Ca2+-stimulated
ATPases
not involved
in Ca2+
transport.
Moreover the ATPase involved in Ca2+ transport by our membrane vesicles is clearly different from
that of skeletal muscle SR because it can utilize only
ATP (Table 5) in contrast to the spectrum of substrates
that can support Ca2+ transport
in the latter (24, 34, 25,
12). Myometrial
membranes resemble heart SR (17) and
membranes
from mesenteric
artery (51) as well as microsomes from several other types of cells (35, 36, 37) in
this regard. The stoichiometry
of Ca2+ transport
by this
ATPase is unknown.
Our procedure requires only 5-6 h from the death of
the rats to the complete fractionation
of the tissue without the need for high salt extraction,
thus allowing
early study of Ca2+ uptake. Preliminary
studies indicated that both SER and mitochondria
lost some Ca2+sequestering
ability after storage. Thus despite appreciable mutual contamination
of endoplasmic reticulum
and mitochondrial
fractions,
new information
was obtained.
Compared to skeletal muscle SR (27), the vesicular
fractions from rat myometrium
exhibited a lower rate of
Ca2+ uptake,
a lower net uptake of Ca2+ at all Ca2+
concentrations
tested, and a much lower enhancement
of Ca2+ transport
by oxalate and a more stringent
substrate requirement.
In addition, SR isolated from rat skeletal muscle by
the same techniques
as used here takes up 5 times as
much Ca2+ in 30 s from a solution of 0.3 PM Ca2+ as PM
does in 10 min under otherwise
identical conditions
(unpublished
studies). This difference is consistent with
the relative rates of relaxation
of these two types of
muscle. However,
the apparent rates of binding and the
maximum
amount of Ca 2+ that can be bound by the
myometrial
fractions might be reduced as a result of
vesicle leakiness and/or the presence of vesicles with the
wrong orientation
with respect to the direction of transport. Nevertheless,
the Ca2+ uptake by the myometrial
vesicular fractions from solutions of high Ca2+ concentrations was similar in magnitude
to that reported for
rabbit aorta (20) and guinea pig intestine
(26). The
results suggest that the mechanisms
of Ca2+ transport of
PM, SER, and RER are similar to each other but different from that of the SR of skeletal muscle.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 16, 2017
activity
of cytochrome
c oxidase present in the mitochondrial
fraction (Table l)* The RER had a similar
cytochrome
c oxidase activity to that of the mitochondrial fraction
and its ATP-dependent
Ca2+ uptake was
inhibited 58% by azide. In the case of both SER and
RER, however,
total ATP-dependent
Ca2+ uptake was
much less than that predicted from the specific activity
of cytochrome c oxidase activity relative to Ca2+ uptake
by the mitochondrial
fraction, There was a large azideinsensitive
component in each case and their Ca2+ uptake properties were also markedly different (Figs. 3,4,
and 5). This suggests that the mitochondrial
contamination of both SER and RER resulted mainly from damaged particles that did not fully contribute
to Caz+ uptake.
Thus much of the material in these fractions
is derived from the endoplasmic
reticulum.
However,
the
lack of an established
marker for smooth muscle endoplasmic reticulum
(50) made it impossible to determine
either the purification
of endoplasmic
reticulum
in
these fractions,
or the contamination
of these fractions
in PM. Morphological
evidence, however,
strongly supports our conclusions that the fraction designated RER
is composed substantially
of material derived from the
rough endoplasmic
ret&&m
of the muscle. An estimate of the amount of endopl .asmic reticulum
in each
fraction might be made from the effect of oxalate on
ATP-dependent
uptake in each fraction. The mitochondrial fraction had an azide-insensitive
ATP-dependent
uptake of about 5 pmol Ca2+ 8-I protein under the standard assay conditions
( see Tab1 e 2) and an increase in
uptake due to oxalate of about 28 ,umol Ca2+ g-‘. The
respective numbers for the RER fraction were 5 and 31,
indicating that there may have been as much material
of rough endoplasmic reticulum
origin in the mitochondrial fraction as there was in RER. If this really was the
case cytochrome c oxidase activities and azide sensiti vities will give an overestimate
in
of the contamination
other fractions due to mitochondria.
However,
there is
no independent evidence that in smooth muscle an “oxalate effect” is unique to the endoplasmic reticulum,
and
the increase with this anion in the mitochondrial
fraction was not statistically
reliable.
The fact that the presence of the ionophorous antibiotics in the incubation
medium lowered the Ca2+ uptake
by PM, SER, and RER to values close to those obtained
in the absence of ATP (Tables 2 and 3) suggests that a
major fraction of the ATP-dependently
accumulated
Ca2+ is transported
across the vesicle membrane
and
that a Ca2+ concentration
gradient is formed in all three
cases. Thus the lack of effect of oxalate in augmenting
Ca2+ uptake by PM (Table 2) appears to be due to a lack
of permeability
of the PM to oxalate rather than to
either the absence of transport
by PM or a higher leakiness of PM vesicles to Ca2+. In this discussion
it is
assumed that these ionophores
act by allowing
Ca2+
diffusion across membranes
(38), but not by inhibiting
Ca2+ binding or exchange (17). Direct evidence that net
uptake rather than exchange of Ca2+ occurred is not yet
available.
The apparent presence of an extra-splitting
of ATP
due to added Ca2+ shown in Table 4 seems to support the
CRANKSHAW,
CA'+
REGULATION
IN
RAT
c57
MYOMETRIUM
being the intracellular
pH and the intracellular
concentrations of MgATP. The ability of the various fractions
to transport
Ca2+ is undoubtedly
dependent upon these
two variables,
and Godt (22) has clearly shown that the
Ca2+ dependence of skinned frog muscle fibres is dependent upon the MgATP concentration.
Thus it is impossible to determine precisely the ranges of Ca*+ concentrations
(or states of contraction)
over which the
various organelles
are most important
for relaxation.
Additionally,
it has been suggested that functional relationships might exist in smooth muscle between closely
associated plasma membrane,
endoplasmic
reticulum,
and mitochondria
(15, 23); such relationships
are necessarily lost in subfractionation
studies of this type.
Since the completion of our study, Moore et al. (35)
found that rough as well as smooth endoplasmic reticulum from rat liver was able to actively take up Ca2+
although the latter took up Ca2+ more rapidly than the
former. It is not surprising
that both types of ER exhibit
this activity because few or no qualitative
functional
differences exist between rough and smooth microsomes
with the exception of the ability to carry out protein
synthesis
(13). Also we have found in sodium dodecyl
sulfate-gel
electrophoresis
studies that the membrane
proteins of the two fractions
from myometrium
show
many similar but some different proteins (A. Murray,
and E. E. Daniel unpublished
studies). Ford and Hess
(21) and Valli&res et al. (47) have recently reported that
mitochondria
from vascular
smooth muscle did not
rapidly take up Ca 2+ from solutions of low Ca2+ activity.
This conclusion is in agreement
with our results
on
myometrial
mitochondria,
Of particular
importance
is
their demonstration
that mitochondria
from vascular
smooth muscle retain and tolerate high Ca2+ loads without loss of oxidative phosphorylation
(47).
In summary the results of our studies provide the first
direct evidence for a role for the plasma membrane in
the regulation
of Ca*+ activity of smooth muscle. Furthermore, they provide independent support for electron
probe X-ray microanalysis
studies (43) which suggest a
potential role for mitochondria
and endoplasmic reticulum in this process.
We thank
Mr.
Gus Duchon
for providing
the electron
micrographs.
The technical
assistance
of Miss Eda Lee and Mr. James
Allan is gratefully
acknowledged.
Hoffman-LaRoche
and Eli Lilly
donated
X-537A
and A23187,
respectively.
This work was supported
by the Alberta
Heart
Foundation
and
the Medical
Research
Council
of Canada.
R. A. Janis was a Postdoctoral
Fellow
of the Canadian
Heart
Foundation.
Present
address:
Dept.
of Physiology,
Northwestern
University
Medical
School, Chicago,
Illinois
60611.
D. J. Crankshaw
was a Predoctoral
Fellow
of the Muscular
Dystrophy
Association
of Canada.
Present address:
Div. of Reproductive
Biology,
McMaster
Medical
Centre,
Hamilton,
Ontario,
Canada
L8S
459
Received
for publication
22 March
1976.
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